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RETRACTED ARTICLE: LncRNA BCRT1 promotes breast cancer progression by targeting miR-1303/PTBP3 axis

RETRACTED ARTICLE: LncRNA BCRT1 promotes breast cancer progression by targeting miR-1303/PTBP3 axis Background: Long noncoding RNAs (lncRNAs) play crucial roles in tumor progression and are aberrantly expressed in various cancers. However, the functional roles of lncRNAs in breast cancer remain largely unknown. Methods: Based on public databases and integrating bioinformatics analyses, the overexpression of lncRNA BCRT1 in breast cancer tissues was detected and further validated in a cohort of breast cancer tissues. The effects of lncRNA BCRT1 on proliferation, migration, invasion and macrophage polarization were determined by in vitro and in vivo experiments. Luciferase reporter assay and RNA immunoprecipitation (RIP) were carried out to reveal the interaction between lncRNA BCRT1, miR-1303, and PTBP3. Chromatin immunoprecipitation (ChIP) and RT-PCR were used to evaluate the regulatory effect of hypoxia-inducible factor-1α (HIF-1α) on lncRNA BCRT1. Results: LncRNA BCRT1 was significantly upregulated in breast cancer tissues, which was correlated with poor prognosis in breast cancer patients. LncRNA BCRT1 knockdown remarkably suppressed tumor growth and metastasis in vitro and in vivo. Mechanistically, lncRNA BCRT1 could competitively bind with miR-1303 to prevent the degradation of its target gene PTBP3, which acts as a tumor-promoter in breast cancer. LncRNA BCRT1 overexpression could promote M2 polarization of macrophages, mediated by exosomes, which further accelerated breast cancer progression. Furthermore, lncRNA BCRT1 was upregulated in response to hypoxia, which was attributed to the binding of HIF-1α to HREs in the lncRNA BCRT1 promoter. Conclusions: Collectively, these results reveal a novel HIF-1α/lncRNA BCRT1/miR-1303/PTBP3 pathway for breast cancer progression and suggest that lncRNA BCRT1 might be a potential biomarker and therapeutic target for breast cancer. Keywords: LncRNA BCRT1, miR-1303, PTBP3, Progression, Breast cancer * Correspondence: qifengy_sdu@163.com Department of Breast Surgery, Qilu Hospital of Shandong University, Jinan, Shandong 250012, People’s Republic of China Pathology Tissue Bank, Qilu Hospital of Shandong University, Jinan, Shandong 250012, People’s Republic of China © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 2 of 20 Background In the present study, we analyzed public microarrays Breast cancer is one of the most common malignancies to screen lncRNAs that are differentially expressed in among women worldwide. Despite advances in diagnosis breast cancer. LncRNA BCRT1 (breast cancer-related and combined treatments, the prognosis of breast cancer transcript 1), which was significantly overexpressed in patients remains unsatisfactory [1, 2]. Metastasis is one breast cancer tissues and associated with poor prognosis of the leading causes of cancer-related death [3], which of breast cancer patients, was selected for further investi- greatly hinders treatment success. Therefore, a more gation. LncRNA BCRT1 functioned as a tumor promoter comprehensive understanding of the mechanism of pro- by competitively binding with miR-1303 to protect PTBP3 gression and metastasis is important for improving the from degradation and thus promoted the growth and pro- prognosis of breast cancer patients. gression of breast cancer cells both in vitro and in vivo. Recently, long noncoding RNAs (lncRNAs) have been Moreover, lncRNA BCRT1 could be transferred to macro- found to be involved in a variety of physiological and phages via exosomes, promoting M2 polarization and en- pathological processes [4, 5], especially in cancers [6]. hancing its effect on tumor progression. Further study LncRNAs are transcripts with more than 200 nucleotides revealed that lncRNA BCRT1 was induced by hypoxia via that have no protein-coding potential [7]. Despite the HIF-1α-dependent transcriptional regulation, which conse- lack of cross-species conservation [8], researchers in our quently facilitated hypoxia-induced EMT. Our results pro- laboratory and others have demonstrated that lncRNAs vide novel insight into the metastatic mechanism of breast are frequently dysregulated in cancers and are involved cancer and a promising therapeutic target for breast cancer in the progression and metastasis of multiple malignan- treatment. cies [9, 10]. LncRNA ANCR was found to mediate the degradation of EZH2 and thus attenuate the metastatic Methods ability of breast cancer [11]. Moreover, lncRNA AGAP2- Patients and specimens AS1 was found to be upregulated in breast cancer and Human breast cancer tissues and corresponding normal tis- was associated with trastuzumab resistance [12]. How- sues were obtained from patients admitted to Qilu Hospital ever, the clinical significance and biological mechanisms from January 2004 to December 2011. All participants of the vast majority of lncRNAs in the regulation of provided written informed consent, and the research was breast cancer remain largely unknown. approved by the Ethical Committee on Scientific Research Several studies have suggested that lncRNAs may func- of Shandong University Qilu Hospital. tion as competing endogenous RNAs (ceRNAs) to regulate the biological functions or expression of microRNAs. For instance, lncRNA LINC00963 promotes tumorigenesis and RNA sequencing analysis radioresistance by acting as a ceRNA for miR-324-3p in Breast cancer gene expression data were downloaded breast cancer cells [13]. LncRNA NONHSAT101069 acted from The Cancer Genome Atlas (TCGA) and the Gene as a ceRNA by effectively sponging miR-129-5p, thereby Expression Omnibus (GEO) dataset GSE112848. The modulating the repression of Twist1 and promoting epiru- data analysis was performed with R software using the bicin resistance, migration, and invasion of breast cancer DEGseq package. The threshold set for significant differ- cells [14]. Previous studies have revealed that hypoxia, a ences was log |fold change| ≥ 1 and P-value < 0.05. major hallmark of the tumor microenvironment, is associ- ated with the progression and metastasis of many solid tu- mors. HIF-1α is an extensively studied hypoxia-inducible Cell culture and reagents factor (HIF) that mediates the cellular response to hypoxia All cell lines were purchased from the American Type through transactivation of downstream target genes [15]. Culture Collection (Manassas, VA) and were cultured ac- Under normoxic conditions, HIF-1α is subjected to prote- cording to the manufacturer’s instructions. MCF10A cells asome degradation, whereas, hypoxic conditions protect were cultured in Dulbecco’smodifiedEagle’smedium HIF-1α from degradation, allowing HIF-1α translocation (Invitrogen, USA) containing 5% horse serum, 10 μg/ml into the nucleus to initiate gene expression [16]. Re- insulin, 20 ng/ml EGF, 100 ng/ml cholera toxin, and cently, the roles of hypoxic conditions in regulating 0.5 μg/ml hydrocortisone. MCF-7, MDA-MB-231, MDA- lncRNA expression have received extensive attention, MB-468, and HEK293T cells were cultured with Dulbec- and various hypoxia-responsive lncRNAs have been co’smodifiedEagle’s medium. T47D and THP1 cells were reported to play important roles in tumorigenesis and cultured with RPMI 1640 medium. The above media con- tumor progression [17]. However, more investigations tained 100 U/ml penicillin, 100 μg/ml streptomycin and should be carried out on the mechanism of hypoxia 10% fetal bovine serum (Invitrogen, USA). The medium in mediating aberrant lncRNA expression as well as for T47D cells also contained 10 μg/ml insulin. All cells the functions of lncRNAs in breast cancer. were cultured in a 5% CO2-humidified incubator at 37 °C. RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 3 of 20 RNA extraction and quantitative real-time PCR analysis curves were determined by calculating the relative value Total RNA was isolated using TRIzol reagent (Invitrogen, of absorbance measured at 570 nm on a microplate USA). Complementary DNA (cDNA) was synthesized reader (Bio-Rad, USA). using a PrimeScript RT reagent kit (TaKaRa, Japan). For miRNAs, reverse transcription was carried out using the Colony formation assay PrimeScript miRNA cDNA Synthesis Kit (TaKaRa, Japan). Transfected cells were counted and seeded at 500 cells RT-PCR was performed using SYBR Premix Ex Taq I. per 6 cm plate. After 10–14 days, cell colonies were Primers used in the study are listed in Additional file 1: washed with PBS, fixed with ethanol for 15 min and Table S1. β-Actin was used as an internal control for stained with crystal violet for 20 min. The colonies were mRNA. U6 was used as an internal control for miRNA. imaged and counted. Relative RNA abundances were calculated by the standard -ΔΔCt 2 method. EdU incorporation assay Transfected cells were seeded into 96-well plates at a Subcellular fractionation density of 1 × 10 cells/well. The EdU incorporation Nuclear and cytoplasmic separation was performed assay kit (RiboBio, China) was used to evaluate cell pro- using the PARIS Kit (Life Technologies, USA) according liferation. A fluorescence microscope (Nikon, Japan) was to the manufacturer’s instructions. used to obtain images. Fluorescence in situ hybridization (FISH) Cell apoptosis assay The FISH assay was performed in MDA-MB-231 cells ac- EDTA-free trypsin was used to collect cells, and the cells cording to the specifications of the manufacturers. The were resuspended in 500 μl of binding buffer. After incu- Cy3-labeled lncRNA BCRT1 probes used in our study bation with 5 μl Annexin V-FITC and 5 μl PI (BD Biosci- were designed and synthesized by GenePharma (Shanghai, ences, USA) for 15 min in the dark, the cells were China). Briefly, the prepared cells were fixed with 4% para- examined on a FACSCalibur (BD, Biosciences, USA) formaldehyde for 30 min. After permeabilization, the cells within 1 h. were incubated with specific probes at 37 °C overnight. The cell nuclei were stained with DAPI (Sigma-Aldrich, Transwell assay USA). The staining results were observed using a fluores- Transwell assays were performed using Transwell cham- cence microscope (Nikon, Japan). bers (pore size 8 μm; Costar Corporation, USA) with or without matrigel (BD Biosciences, USA). A total of 1 × Plasmid construction and transfection 10 cells were added to the upper insert. The lower The full-length lncRNA BCRT1 cDNA was cloned into chamber contained 700 μl medium with 20% FBS as a pcDNA3.1 (Invitrogen, USA). The primers used for vector chemoattractant. After incubation for 24–48 h, the cells construction are showed in Additional file 1:Table S1.The on the lower surface were fixed with ethanol and stained lncRNA BCRT1 plasmid and corresponding empty vector with 0.2% crystal violet. The relative cell number was were transfected into breast cancer cells using Lipofecta- calculated. mine 2000 reagent (Invitrogen, USA). G418 (2 mg/ml) was used to generate stably transfected cells. For PTBP3 knock- Tube formation assay down, the pLKO.1 plasmid was used as a negative control. Seventy-five microliters of Matrigel (BD Biosciences, The 3′UTR sequences of lncRNA BCRT1 and PTBP3 with USA) was pipetted into each well of a 48-well plate and wild-type or mutant miR-1303 binding sites were cloned allowed to solidify for more than 1 h at 37 °C. HUVECs into the pmirGLO vector (Invitrogen, USA). Different frag- were suspended in the indicated conditioned medium ments of the lncRNA BCRT1 promoter were cloned into and seeded onto the gel. After 4–6 h of incubation, a the pmirGLO vector. The negative control, lncRNA BCRT1 bright-field microscope was used to observe the tubular siRNAs, and miR-1303 mimics (GenePharma, China) were structures and acquire images. Tube formation was transfected using Lipofectamine 2000. quantified by measuring the total length of the tubes using ImageJ software. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay Western blot assay Transfected cells were seeded at a density of 5000 cells/ Cell proteins were extracted and separated by 10% SDS- well in 96-well plates. After incubation, 20 μl of 5 mg/ml PAGE gels and transferred to 0.22 μm PVDF membranes MTT was added to each well and incubated for another (Millipore, USA). The membranes were blocked with 5% 4 h. Then, the supernatants were carefully removed, and skim milk powder and incubated with specific antibodies 100 μl DMSO was added to each well. The proliferation at 4 °C overnight. The membranes were then incubated RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 4 of 20 with the appropriate secondary antibodies, and an ECL Antibodies used for the RIP assay included anti-AGO2 detection system (Bio-Rad, USA) was used to detect the and control IgG (Millipore, USA), and the coprecipitated protein bands. β-Actin was used as a control. The primary RNAs were used for cDNA synthesis and evaluated by antibodies and secondary antibodies used are described in qRT-PCR. Additional file 2: Table S2. Isolation and characterization of exosomes Tumor xenograft model Exosomes were isolated from the supernatant of MDA- MDA-MB-231 cells (1 × 10 cells) with or without MB-231 cells that had been cultured in DMEM containing lncRNA BCRT1 overexpression were suspended in 10% exosome-depleted FBS for 48 h via a polyethylene 200 μl PBS and subcutaneously injected into each flank glycol-based method as previously described [18]. Briefly, of 4–6-week-old BALB/c nu/nu female mice. The mice the culture medium was centrifuged at 500×g for 5 min, were sacrificed after 30 days, and the maximum (L) and and the supernatant was further centrifuged at 2000×g for minimum (W) length and weight of the tumors were 30 min. Then, 2× PEG solution was added to the super- measured. Tumor volume was calculated as ½LW .To natant and gently mixed. The mixture was stored at 4 °C evaluate the influence of lncRNA BCRT1 on metastasis, formore than12h andthen centrifuged at 10,000×g for 1 5×10 cells were injected into the lateral tail veins of h at 4 °C to collect the exosomes. The supernatant was re- nude female mice (five mice per group). After 4 weeks, moved, and the exosome pellet was resuspended in 0.2 μm- the mice were euthanized, and the lungs were collected filtered PBS. to evaluate the number of pulmonary metastatic lesions. Hematoxylin and eosin (H&E) staining was performed Exosome uptake assay for tissue morphology evaluation. The animal experi- PKH26, a red fluorescent dye (Sigma-Aldrich, USA), was ments were approved by the Shandong University Ani- used to label exosomes obtained from conditioned mal Care and Use Committee. medium [19]. After incubation with the recipient cells for 12 h, fluorescence microscopy was used for imaging. Immunohistochemical (IHC) analysis The paraffin-embedded sections were dewaxed in xylene Kaplan-Meier plotter tool analysis and rehydrated in alcohol. Endogenous peroxidase was The Kaplan-Meier Plotter tool (http://kmplot.com/analysis/) blocked by 3% H O , and microwave heating was per- 2 2 was used to determine the association between PTBP3 and formed for antigen retrieval. After blocking nonspecific the prognosis of breast cancer patients. antigen binding with 5% BSA at 37 °C for 1 h, the sections were incubated with a specific primary antibody against Chick chorioallantoic membranes (CAM) Ki67, PTBP3 or CD31 (1100 dilution, Abcam, USA) at The fertilized chicken eggs were cultured at 37 °C in an 4 °C overnight. After incubating with the corresponding 80% humidified atmosphere for 7 days. Then, a square secondary antibodies at 37 °C for 1 h, the sections were window was cut on the shell to expose the CAM and was stained with diaminobenzidine and counterstained with covered with a gelatin sponge (0.3 cm × 0.3 cm × 0.3 cm) hematoxylin. Representative images were taken using an containing PBS or the indicated conditioned medium Olympus light microscope. (CM). Next, tape was used to cover the window for further incubation. After 2 days, the CAM were visualized under Luciferase assay astereoscope. The wild-type or mutant lncRNA BCRT1 or 3’UTR of PTBP3 was amplified and cloned into pmirGLO vector Chromatin immunoprecipitation (ChIP) separately. Then, HEK293T cells were plated on a 96- ChIP assays were performed using a ChIP kit (CST, well plate and cotransfected with wild-type or mutant USA) following the manufacturer’s instructions. Briefly, luciferase plasmids and miR-1303 or control miRNA. cells were crosslinked with formaldehyde and sonicated The lncRNA BCRT1 promoter segment was cloned into to an average length of 200–1000 bp. Immunoprecipita- the pGL3-basic vector. The pGL3-BCRT1 and pRL-TK tion was conducted with an anti-HIF-1α antibody vectors were cotransfected with si-NC or si-HIF1α.A (Abcam, UK) or IgG control. Precipitated DNA was Dual-Luciferase Reporter Assay System (Promega, USA) amplified by RT-PCR. Primer sequences are provided in was used to measure the luciferase activity. Supplementary Table S4. RNA immunoprecipitation (RIP) assay A Magna RIP RNA-Binding Protein Immunoprecipita- Elisa tion Kit (Millipore, USA) was used to determine the re- The TGFβ concentration in the cell culture medium was lationship between lncRNA BCRT1 and miR-1303. measured by ELISA using the Quantikine human TGFβ RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 5 of 20 ELISA kit (R&D Systems, USA) according to the manu- expression levels were correlated with significantly facturer’s instructions. shorter disease-free survival (DFS) and overall survival (OS) (Fig. 1e). Univariate (Additional file 5: Table S4) Statistical analysis and multivariate (Additional file 6: Table S5) analyses Data are expressed as the mean ± S.D. of three inde- further showed that lncRNA BCRT1 expression was a pendent experiments and analyzed by the SPSS software major prognostic factor for breast cancer patients. The program (version 17.0). Student’s t-test was used for results of nuclear/cytoplasmic RNA fractionation from two-group comparisons. Kaplan–Meier survival analysis the subcellular distribution assay confirmed that lncRNA was performed for survival rate calculation. Cox propor- BCRT1 was mainly located in the cytoplasm (Fig. 1f), tional hazards model multivariate analyses were used to which was further confirmed by the fluorescence in situ evaluate the significance of lncRNA BCRT1 expression hybridization (FISH) analysis (Fig. 1g). Collectively, these and clinicopathological features on overall survival. P < findings revealed that lncRNA BCRT1 was upregulated 0.05 was considered statistically significant. in breast cancer and that high expression of lncRNA BCRT1 was associated with poor outcomes in breast Results cancer. LncRNA BCRT1 expression is upregulated in breast cancer and associated with poor prognosis LncRNA BCRT1 promotes cell proliferation and tumor To identify important lncRNAs that potentially participate growth in breast cancer in breast cancer progression, we analyzed the lncRNA ex- To determine the biological function of lncRNA BCRT1 in pression profiles using public databases (GSE112848 and a breast cancer cells, short interference siRNAs against hu- TCGA dataset) (Fig. 1a-b). In the present study, we mainly man lncRNA BCRT1 (si-BCRT1) were applied to knock focused on the upregulated lncRNAs given that these down lncRNA BCRT1, and the knockdown efficiency was lncRNAs might serve as therapeutic targets or prognostic confirmed by RT-PCR (Fig. 2a and Additional file 7:Figure biomarkers. Among them, lncRNA BCRT1 (breast cancer S2a). After lncRNA BCRT1 knockdown, the proliferation, related transcript 1), which was one of the prominently colony-formation abilities, and DNA synthesis activities of upregulated lncRNAs in breast cancer tissues, was chosen breast cancer cells were significantly decreased (Fig. 2b-d for further evaluation. LncRNA BCRT1 is located on and Additional file 7: Figure S2b). The results of flow cy- 10q25.1 in humans and is composed of 3 exons with a full tometry revealed that lncRNA BCRT1 knockdown obvi- length of 1013 nt (Additional file 3:Figure S1a). These- ously increased the total apoptosis rate in breast cancer quence of full-length lncRNA BCRT1 and its secondary cells (Fig. 2e). On the other hand, when lncRNA BCRT1 structure based on minimum free energy (MFE) are was overexpressed by transfection with the pcDNA3.1 plas- shown in Additional file 3: Figure S1b and c, respectively. mid containing the lncRNA BCRT1 sequence, the prolifera- Moreover, using the Open Reading Frame (ORF) Finder tion and colony formation of breast cancer cells was and conserved domain database, we found that lncRNA significantly increased (Fig. 2f-g and Additional file 7: BCRT1 had little potential to code proteins, which was in Figure S2c-f). Furthermore, a subcutaneous xenograft accordance with the results of five different online metrics model was used to validate the biological function of (Additional file 3: Figure S1d-f). In addition, we failed to lncRNA BCRT1 in vivo. Consistent with the results identify a valid Kozak consensus sequence in lncRNA in vitro, lncRNA BCRT1 overexpression significantly in- BCRT1, further supporting the notion that lncRNA creased tumor weight and tumor volume compared with BCRT1 had no protein-coding potential [20]. those in the control group (Fig. 2h-i). Moreover, immuno- Compared with that in normal breast epithelial cells histochemistry (IHC) assays confirmed that lncRNA (MCF10A), the expression of lncRNA BCRT1 in four BCRT1 overexpression caused increased Ki67 expression breast cancer cell lines was significantly higher (Fig. 1c). (Fig. 2j), indicating enhanced cell proliferation. Our findings Moreover, we further investigated the lncRNA BCRT1 indicated that lncRNA BCRT1 could promote breast cancer expression levels in 18 paired breast cancer tissues and cell proliferation both in vitro and in vivo. normal breast tissues using real-time PCR analysis, and the results revealed that lncRNA BCRT1 was signifi- LncRNA BCRT1 promotes cell mobility and tumor cantly overexpressed in breast cancer tissues compared metastasis in breast cancer with adjacent normal tissues (Fig. 1d). The association We then investigated the role of lncRNA BCRT1 in the between the clinicopathological characteristics of breast motility of breast cancer cells. The results showed that cancer patients and lncRNA BCRT1 expression level is lncRNA BCRT1 knockdown significantly impaired the summarized in Additional file 4: Table S3. LncRNA migration and invasion of breast cancer cells, whereas BCRT1 was overexpressed in breast cancer tissues with lncRNA BCRT1 overexpression led to increased cell mo- distant metastasis (Fig. 1d), and higher lncRNA BCRT1 bility (Fig. 3a-b and Additional file 7: Figure S2g-h). RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 6 of 20 Fig. 1 LncRNA BCRT1 upregulation is associated with advanced progression and poor prognosis in breast cancer. a Heat maps showing the top differentially expressed lncRNAs in breast cancer samples compared to normal tissues (left, GSE112848; right, TCGA). The red shades represent high expression, and green shades represent low expression. b Volcano plots showing the expression profiles of lncRNAs. c-d RT-PCR analysis was used to detect the expression of lncRNA BCRT1 in cell lines and tissues. Actin was the internal control. e Kaplan–Meier analysis showed the association between lncRNA BCRT1 expression and disease-free survival or overall survival of breast cancer patients (n = 68). f The expression level of lncRNA BCRT1 in the subcellular fractions of MDA-MB-231 cells was detected by qRT-PCR. U6 and GAPDH were used as nuclear and cytoplasmic markers, respectively. g The location of lncRNA BCRT1 (red) in MDA-MB-231 cells was determined by FISH assay. DAPI-stained nuclei are blue. (**P < 0.01 and ***P < 0.001) Moreover, we used breast cancer-conditioned medium to length (Fig. 3c). Given that epithelial-mesenchymal transi- stimulate angiogenesis in HUVECs to evaluate angiogenesis tion (EMT) is one of the major mechanisms for cancer me- activity in vitro [21]. The results showed that the relative tastasis, we further evaluated the effect of lncRNA BCRT1 length of tubes was decreased in the si-BCRT1 group com- on EMT-related markers. Western blot analysis showed pared with the si-NC group. On the other hand, lncRNA that lncRNA BCRT1 knockdown could increase the expres- BCRT1overexpressionled to asignificantlyelevatedtube sion of epithelial markers (E-cadherin) and decrease the RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 7 of 20 Fig. 2 LncRNA BCRT1 knockdown inhibited breast cancer cell proliferation in vitro and in vivo. a The expression levels of lncRNA BCRT1 in MDA- MB-231 and MDA-MB-468 cells after transfection with si-NC or si-BCRT1 were detected by RT-PCR. b-c The effects of lncRNA BCRT1 knockdown on the proliferation of MDA-MB-231 and MDA-MB-468 cells were examined by MTT assay (b) and colony formation assays (c). Experiments were performed in triplicate. d EdU assays were used to detect the proliferation rate of MDA-MB-231 and MDA-MB-468 cells after lncRNA BCRT1 knockdown. Columns are the average of three independent experiments. e Flow cytometry was performed to determine the effect of lncRNA BCRT1 on apoptosis by flow cytometry analysis. f RT-PCR was used to determine the efficiency of the lncRNA BCRT1-overexpressing vector. g MTT assay indicated an increased proliferative ability of MDA-MB-231 and MDA-MB-468 cells after lncRNA BCRT1 overexpression. h MDA-MB-231 cells were stably transfected with the lncRNA BCRT1-overexpressing vector or control vector and injected subcutaneously into nude mice. Compared with the vector group, lncRNA BCRT1 overexpression promoted tumor growth. i Tumor volume and weight were significantly increased in the lncRNA BCRT1-overexpressing group. j Representative images of H&E and Ki67 staining in the tumor. Immunohistochemical staining revealed that lncRNA BCRT1 overexpression led to increased expression of Ki67. (**P < 0.01 and ***P < 0.001) RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 8 of 20 Fig. 3 (See legend on next page.) RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 9 of 20 (See figure on previous page.) Fig. 3 LncRNA BCRT1 knockdown inhibited breast cancer cell metastasis in vitro and in vivo. a Transwell migration and invasion assays were used to evaluate the motility of MDA-MB-231 and MDA-MB-468 cells transfected with si-NC or si-BCRT1. Columns are the average of three independent experiments. b LncRNA BCRT1 overexpression led to increased migration and invasion of MDA-MB-231 and MDA-MB-468 cells. c Tube formation in HUVECs was inhibited by conditioned medium from MDA-MB-231 cells transfected with si-BCRT1 and was promoted by that from MDA-MB-231 cells transfected with the lncRNA BCRT1-overexpressing vector. d EMT-related proteins were detected by western blot in MDA-MB-231 and MDA-MB-468 cells after knockdown or overexpression of lncRNA BCRT1. e Stably transfected MDA-MB-231 cells were injected into the tail veins of nude mice (n = 5). Representative images of lungs and H&E staining of lungs isolated from mice. LncRNA BCRT1 overexpression resulted in an increased number of lung metastatic colonies. f Representative immunohistochemistry staining of CD31 in the indicated xenografts. The corresponding statistical plots are presented in the lower panel. (**P < 0.01 and ***P < 0.001) expression of mesenchymal markers (such as Fibronec- breast cancer. A negative association between lncRNA tin, N-cadherin, and Vimentin) (Fig. 3d), indicating that BCRT1 and miR-1303 was also detected in xenograft tu- lncRNA BCRT1 could regulate the EMT process to mors (Additional file 8:FigureS3b). modulate breast cancer progression. To confirm these Then, we examined the role of miR-1303 in breast can- findings in vivo, we injected breast cancer cells through cer. Higher expression of miR-1303 was correlated with the tail vein to establish a pulmonary metastasis model better overall survival of breast cancer patients according to in nude mice. Two of the five mice (2/5) injected with the LinkedOmics database [24](Additional file 8:Figure breast cancer cells in the control group and all five S3c), indicating that miR-1303 acted as a tumor suppressor mice (5/5) injected with breast cancer cells in the in breast cancer. The transfection efficiency of miR-1303 lncRNA BCRT1-overexpressing group showed meta- mimics was determined by RT-PCR (Fig. 4eand Additional static foci in their lungs after 4 weeks (Fig. 3e). Then, file 8: Figure S3d), and miR-1303 overexpression led to a all mice were sacrificed, and their lungs were subjected decreased proliferation rate and increased apoptotic rate of to hematoxylin and eosin (H&E) staining. The results breast cancer cells (Fig. 4f-g, and Additional file 8:Figure revealed that lncRNA BCRT1 overexpression remark- S3e). Moreover, miR-1303 overexpression decreased cell ably increased the volume and number of lung meta- migration and invasion (Fig. 4h and Additional file 8:Figure static lesions compared with those in the control group S3f).Importantly,rescueexperiments further validated the (Fig. 3f). Similarly, vascular density was increased in the functional relationship between lncRNA BCRT1 and miR- lncRNA BCRT1-overexpressing group (Fig. 3f). Taken 1303 (Fig. 3i-k). Moreover, lncRNA BCRT1 expression was together, these data show that lncRNA BCRT1 pro- decreased after miR-1303 overexpression in breast cancer motes tumor metastasis in breast cancer cells. cells (Additional file 8: Figure S3g), indicating a reciprocal suppression between them. Overall, we chose miR-1303 as LncRNA BCRT1 functions as a miR-1303 sponge in breast an inhibitory target of lncRNA BCRT1 for further investiga- cancer cells tion in breast cancer. Recently, many lncRNAs have been reported to function as competing endogenous RNAs (ceRNAs) in modulating the LncRNA BCRT1 upregulates PTBP3 expression via expression and biological functions of miRNAs [22, 23]. inhibition of miR-1303 Since lncRNA BCRT1 was distributed predominantly in Using the miRDB, miRWalk, miRPathDB, and TargetS- the cell cytoplasm, we hypothesized that lncRNA BCRT1 can databases, we found that PTBP3 was a potential tar- might act as a miRNA sponge to prevent miRNAs from get of miR-1303 (Fig. 5a). Additionally, we found that binding with their target mRNAs. Through the RegRNA the expression of PTBP3 was elevated in breast cancer database, we identified miR-1303 as a potential target of tissues compared to normal tissues using the TCGA and lncRNA BCRT1 (Fig. 4a). To validate the binding potential, GEO databases (Fig. 5b), and high PTBP3 expression a luciferase reporter assay was performed. Overexpression was associated with poor prognosis of breast cancer pa- of miR-1303 significantly reduced the luciferase activity of tients (Additional file 9: Figure S4). Furthermore, we the pmirGLO-BCRT1-wt vector but failed to decrease that found that the expression of PTBP3 was positively asso- of the mutant vector (Fig. 4b). The AGO2 immunoprecipi- ciated with the expression of lncRNA BCRT1 in breast tation assay showed that the AGO2 antibody was able to cancer cells (Fig. 5c). Therefore, PTBP3 was selected as pull down both endogenous lncRNA BCRT1 and miR- a putative target of miR-1303 for further observation. 1303 (Fig. 4c), further validating their binding potential. Luciferase assays showed that overexpression of miR- Moreover, lncRNA BCRT1 knockdown promoted miR- 1303 decreased the luciferase activity of the wild-type 1303 expression (Fig. 4d), whereas lncRNA BCRT1 overex- PTBP3 reporter but not the mutant reporter (Fig. 5d), pression inhibited miR-1303 expression (Additional file 8: indicating that PTBP3 was the direct target of miR-1303. Figure S3a). Our above data supported the hypothesis that Furthermore, the mRNA and protein levels of PTBP3 miR-1303 is an inhibitory target of lncRNA BCRT1 in were reduced by miR-1303 overexpression (Fig. 5e) or RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 10 of 20 Fig. 4 LncRNA BCRT1 acts as a sponge of miR-1303 in breast cancer. a Schematic diagram representing the predicted binding sites for miR-1303 in lncRNA BCRT1 and mutant sequences of the potential miR-1303 binding sites. b Luciferase assays in HEK293T cells cotransfected with wild-type or mutant lncRNA BCRT1 and miR-1303 or NC. The data are shown as the means ± SD of triplicate samples. c Anti-AGO2 RIP was performed in HEK293T cells, followed by RT-PCR to detect the expression of lncRNA BCRT1 or miR-1303 associated with AGO2. d RT-PCR was used to detect the effect of lncRNA BCRT1 knockdown on the expression of miR-1303 in breast cancer cells. e The overexpression of miR-1303 in breast cancer cells was validated by RT-PCR. f The proliferation of breast cancer cells transfected with NC or miR-1303 was measured by MTT assay. g MDA-MB- 231 and MDA-MB-468 cells were transfected with miR-1303 mimics or NC, and the apoptotic rates were determined by FACS analysis. Representative results are shown, and data are presented as the mean ± SD. h Transwell assays were used to measure the migration of breast cancer cells transfected with miR-1303 mimics or NC. i The effects of lncRNA BCRT1 and miR-1303 cotransfection on cell proliferation were measured by MTT assay. j Transwell assay was used to determine the migration of breast cancer cells cotransfected with lncRNA BCRT1 and miR- 1303. k Overexpression of miR-1303 inhibited the effect of conditioned medium from lncRNA BCRT1-overexpressing cells on the tube formation of HUVECs. (*P < 0.05, **P < 0.01, and ***P < 0.001) RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 11 of 20 Fig. 5 LncRNA BCRT1 promoted breast cancer cell proliferation and progression by protecting PTBP3 from miR-1303-induced degradation. a Schematic illustration showing the overlapping target genes of miR-1303 predicted by miRDB, miRWalk, miRPathDB, and TargetScan. b The expression of PTBP3 was increased in breast cancer tissues compared to normal tissues based on the TCGA and GEO databases. c RT-PCR revealed a positive correlation between lncRNA BCRT1 expression and PTBP3 expression in breast cancer cells. d The upper schematic diagram represents the construction of the luciferase reporter plasmids. The lower panel shows the predicted and the mutated binding sites of miR-1303 in the 3′UTR of PTBP3. The statistical graphs on the right show the luciferase activity in HEK293T cells with or without miR-1303 overexpression and transfected with the WT or MUT luciferase plasmids. e RT-PCR and western blot assays revealed the effect of miR-1303 on PTBP3 expression. f RT-PCR and western blot assays showed that lncRNA BCRT1 knockdown repressed the expression of PTBP3. g RT-PCR and western blot assays were used to determine the PTBP3 expression level in MDA-MB-231 cells cotransfected with pcDNA3.1-BCRT1 and miR-1303 mimics. h RT-PCR was used to detect the efficiency of PTBP3 knockdown in breast cancer cells. i MTT assay was performed to examine the proliferation ability after PTBP3 knockdown. j PTBP3 knockdown led to increased cell apoptosis. k Transwell assays revealed that PTBP3 knockdown inhibited the migration and invasion abilities of breast cancer cells. (*P < 0.05, **P < 0.01, and ***P < 0.001) RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 12 of 20 lncRNA BCRT1 knockdown (Fig. 5f). In the rescue ex- attempted to investigate the mechanism mediating the periments, overexpression of miR-1303 could partly communication between breast cancer cells and macro- counteract the corresponding increases in PTBP3 ex- phages. Various studies have reported that lncRNAs can be pression induced by lncRNA BCRT1 overexpression in transferred by exosomes to modulate the tumor micro- breast cancer cells (Fig. 5g). In addition, lncRNA BCRT1 environment [31]. To investigate whether lncRNA BCRT1 overexpression also led to increased expression of can be packed into exosomes, we extracted exosomes from PTBP3 in xenograft tumors (Additional file 10: Figure the cultured supernatants of breast cancer cells and used S5a-b). Previous studies reported that PTBP3 acted as a western blotting to detect the expression of exosome- tumor promoter in various cancers, such as gastric can- related proteins, such as CD63, HSP70, and HSP90 (Fig. cer [25], hepatocellular carcinoma [26], and colorectal 6f). LncRNA BCRT1 overexpression in MDA-MB-231 cells cancer [27]. However, the role of PTBP3 in breast cancer led to increased levels of lncRNA BCRT1 in the secreted has not been fully elucidated. PTBP3 knockdown re- exosomes, whereas lncRNA BCRT1 knockdown produced sulted in significantly inhibited cell proliferation and in- the opposite results (Fig. 6g), indicating the existence of creased cell apoptosis (Fig. 5h-j). Moreover, Transwell lncRNA BCRT1 in exosomes. We labeled MDA-MB-231 assays showed that PTBP3 knockdown led to attenuated cell-derived exosomes with PKH26 and incubated them migration and invasion of breast cancer cells (Fig. 5k). with macrophages to examine exosome incorporation and These data suggested that PTBP3 acted as a tumor pro- confirmed that the labeled exosomal RNAs could be inter- moter in breast cancer, and lncRNA BCRT1 played signifi- nalized by macrophages (Fig. 6h). Then, we cocultured cant roles in regulating PTBP3 expression by regulating unpolarized macrophages with exosomes isolated from miR-1303. lncRNA BCRT1-overexpressing or control MDA-MB-231 cells. The expression of lncRNA BCRT1 and M2 pheno- Exosomal lncRNA BCRT1 promotes M2 phenotype type markers (CD206 and MRC-2) was significantly in- polarization and enhances macrophage-induced tumor creased in the lncRNA BCRT1-overexpressing group progression compared to the control group (Fig. 6i), indicating that Previous studies have reported that tumor-associated mac- exosomal lncRNA BCRT1 promoted M2 polarization. rophages (TAMs), which are considered to have an M2- Then, we investigated the role of lncRNA BCRT1 in like phenotype, are the most abundant immune-related modulating the behaviors of macrophages. As expected, cells in the tumor microenvironment (TME) and partici- supernatants from lncRNA BCRT1-overexpressing cells pate in tumor development by mediating angiogenesis, me- led to increased migration ability of macrophages and tastasis, and immune escape [28–30]. To investigate showed enhanced chemotaxis (Additional file 11: Figure whether lncRNA BCRT1 contributes to M2 polarization, S6a-b). Moreover, supernatant or exosomes from we evaluated the expression of lncRNA BCRT1, M1 lncRNA BCRT1-overexpressing cells promoted the markers, and M2 markers in unpolarized macrophages, expression and secretion of TGF-β compared with the LPS/INF-γ-induced M1 macrophages, and IL-4/IL-13-in- control groups (Additional file 11:FigureS6c-e). To duced M2 macrophages. The results revealed that the ex- further investigate whether lncRNA BCRT1-educated pression levels of M1-associated genes (CD80, MCP-1, M2 phenotype macrophages have the characteristic iNOS, and IL-6) were significantly upregulated in M1 mac- function of tumor promotion, we treated macrophages rophages, whereas those of M2-associated genes, including with exosomes or supernatants isolated from lncRNA CD206 and MRC-2, were significantly upregulated in M2 BCRT1-overexpressing or control cells. Then, the macrophages (Fig. 6a), indicating the successful conditioned medium of educated macrophages was col- polarization of monocytes. Moreover, lncRNA BCRT1 ex- lected and used to treat breast cancer cells or HUVECs. pression was elevated in M2 macrophages compared to The results showed that macrophages treated with exo- M1 macrophages (Fig. 6b), indicating a potential role of somes or supernatants isolated from lncRNA BCRT1- lncRNA BCRT1 in macrophage polarization. After PMA overexpressing groups significantly promoted cell migra- treatment for 24 h, THP-1 cells were transfected with si- tion and angiogenesis (Fig. 6j-k). Moreover, a chick NC or si-BCRT1, and then IL-4 and IL-13 were added for chorioallantoic membrane (CAM) assay revealed that 24 h to induce the M2 phenotype. The results showed that chick embryos injected with conditioned medium of M1 markers were significantly increased, while M2 educated macrophages treated with exosomes or superna- markers were remarkable decreased in the si-BCRT1 group tants isolated from lncRNA BCRT1-overexpressing (Fig. 6c). Accordingly, lncRNA BCRT1 overexpression led groups had an increase in new vessel density (Fig. 6l). to the opposite results (Fig. 6d). Moreover, the supernatant Taken together, these results suggested that lncRNA from lncRNA BCRT1-overexpressing MDA-MB-231 cells BCRT1 could be transferred through exosomes, thus caused an elevated expression of M2 markers compared to promoting M2 phenotype polarization and enhancing its that from control MDA-MB-231 cells (Fig. 6e). Then, we tumor promoting function. RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 13 of 20 Fig. 6 LncRNA BCRT1 could be secreted by breast cancer cells and promoted M2 polarization. a RT-PCR was used to detect the expression of M1 markers and M2 markers after LPS/INF-γ or IL-4/IL-13 treatment. b The expression of lncRNA BCRT1 was elevated in M2 macrophages. c M1 markers (CD80, MCP-1, iNOS, and IL-6) were significantly increased, while M2 markers (CD206 and MRC-2) were remarkably decreased in the lncRNA BCRT1 knockdown group. d LncRNA BCRT1 overexpression led to decreased expression of M1 markers and increased expression of M2 markers. e Conditioned medium derived from lncRNA BCRT1-overexpressing cells further increased the expression of M2 markers and lncRNA BCRT1 in macrophages. f Western blotting analysis of the exosomal markers CD63, Hsp70 and Hsp90 in exosomes derived from breast cancer cells with or without lncRNA BCRT1 overexpression. g Agarose gel electrophoresis and RT-PCR assays were used to detect the expression of lncRNA BCRT1 in exosomes. h Representative microscopy showing the uptake of PKH26-labeled exosomes (red fluorescent dye) derived from MDA-MB-231 cells by recipient macrophages. i The expression of M2 markers and lncRNA BCRT1 in macrophages was detected after culture with the indicated exosomes. j Cell migration was increased after cultured with lncRNA BCRT1-overexpressing exosomes or conditioned media. k-l Tube formation or CAM assays were used to evaluate the angiogenesis ability after culture with lncRNA BCRT1-overexpressing exosomes or conditioned media. (*P < 0.05, **P < 0.01, and ***P < 0.001) LncRNA BCRT1 is transcriptionally regulated by HIF-1α responsible for the aberrant expression of some under hypoxic conditions lncRNAs [32, 33]. To investigate whether lncRNA Hypoxia is one of the major intratumor characteristics BCRT1 is a hypoxia-sensitive lncRNA, breast cancer in various cancers, and several studies have revealed that cells were treated with hypoxia or normoxia for 48 h. the hypoxic microenvironment of cancers might be The results showed that the expression of lncRNA RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 14 of 20 BCRT1 was clearly elevated along with the increase in HIF-1α knockdown (Fig. 8a-c, Additional file 12: Figure HIF-1α expression (Fig. 7a-b). HIF-1α knockdown dra- S7a-c). Previous studies revealed a close association be- matically decreased HIF-1α and lncRNA BCRT1 expres- tween hypoxia and EMT; therefore, the role of lncRNA sion under both normoxic and hypoxic conditions (Fig. BCRT1 in hypoxia-induced EMT was further investi- 7c-e). Moreover, knockdown of HIF-1α substantially at- gated. After treatment with hypoxia, MDA-MB-231 cells tenuated hypoxia-induced lncRNA BCRT1 upregulation demonstrated a more fibroblast-like morphology and el- (Fig. 7c-e). To elucidate the potential mechanism of evated migration ability, which was dramatically reversed hypoxia-induced upregulation of lncRNA BCRT1, we ana- by knockdown of HIF-1α or lncRNA BCRT1 (Fig. 8d-f, lyzed the JASPAR database [34], and two putative HIF-1α Additional file 12: Figure S7d-e). Moreover, the siHIF-1α- response elements (HREs) in the lncRNA BCRT1 pro- repressed EMT profile under hypoxic conditions was moter were identified (Fig. 7f-g). To determine whether obviously rescued by overexpression of lncRNA BCRT1 HIF-1α regulates the expression of lncRNA BCRT1 (Fig. 8d-f, Additional file 12: Figure S7d-e). These results through these HREs, we constructed two luciferase re- indicated that lncRNA BCRT1 might participate in porter vectors containing the full-length lncRNA BCRT1 hypoxia-induced biological functions in breast cancer cells. promoter (HRE1 and HRE2) and a truncated fragment (HRE2). As expected, hypoxia treatment significantly in- Discussion creased the luciferase activity in cells transfected with the The therapeutic methods available to breast cancer pa- full-length lncRNA BCRT1 promoter vector compared tients with metastatic lesions are complicated, but their with the control cells, whereas the lack of HRE1 impaired clinical outcome is less than satisfactory. It is of great the luciferase activity, which suggested that HRE1 was importance to comprehensively understand the molecu- crucial for lncRNA BCRT1 transcription (Fig. 7h). In lar mechanisms involved in breast cancer metastasis and addition, HIF-1α knockdown reversed the luciferase activ- identify novel prognostic predictors. Recently, aberrant ity induced by hypoxia treatment (Fig. 7 h), suggesting that expression of lncRNAs has been reported in various can- hypoxia promoted lncRNA BCRT1 transcription through cers [9, 36], and lncRNAs have been shown to play im- HIF-1α by binding to HRE1 in its promoter region. We portant roles in tumor progression. Increasing studies performed chromatin immunoprecipitation (ChIP) assays have focused on the functions and regulation of lncRNAs with a HIF-1α antibody to further confirm the binding of to discover novel targets for the diagnosis and treatment HIF-1α with the two predicted HREs in the lncRNA of cancers. In this study, we determined that the unchar- BCRT1 promoter (Fig. 7i), and the results confirmed that acterized lncRNA BCRT1 was significantly increased in HRE1 in the lncRNA BCRT1 promoter was the major re- breast cancer tissues compared to normal tissues, and gion mediating HIF-1α-induced transcriptional regulation. high lncRNA BCRT1 expression was associated with poor Using the ChIPBase database, we found that the expres- prognosis of breast cancer patients. Functional studies re- sion of PTBP3 was positively associated with HIF-1α ex- vealed that lncRNA BCRT1 could promote the prolifera- pression (Fig. 7j). Moreover, hypoxia treatment led to tion and mobility of breast cancer cells in vitro and elevated expression of PTBP3 at the mRNA and protein in vivo, indicating a tumor-promoter role in breast cancer. levels (Fig. 7k), while HIF-1α knockdown attenuated this Although several dysregulated lncRNAs have also been effect (Fig. 7l). These results suggested that hypoxia tran- identified, more studies are needed to elucidate their scriptionally regulated lncRNA BCRT1 expression by function. HIF-1α through direct binding with HRE1 on its The biological function of lncRNAs is largely dependent promoter. on their subcellular localization. Accumulated evidence has shown that lncRNAs located in the cytoplasm could LncRNA BCRT1 mediates hypoxia-induced malignant participate in gene regulation at the posttranscriptional properties of breast cancer cells level, including by acting as ceRNAs and protecting the Hypoxia is a hallmark of the tumor microenvironment target mRNAs from repression [37, 38]. By using cell cyto- and is associated with proliferation, metastasis, and drug plasmic/nuclear fractionation and RNA FISH assays, we resistance in various solid tumors [35]. Therefore, we found that lncRNA BCRT1 was preferentially localized in first investigated whether lncRNA BCRT1 was involved the cytoplasm, indicating its potential for functioning as a in hypoxia-induced cell proliferation. Hypoxia treatment miRNA sponge. Subsequently, bioinformatics analysis in- led to increased expression of lncRNA BCRT1 and dicated that there existed binding sites of miR-1303 in the PTBP3, in accordance with enhanced cell proliferation lncRNA BCRT1 sequence, which was further validated by (Fig. 8a-c, Additional file 12: Figure S7a-c). Moreover, luciferase reporter assay and RIP assay. Moreover, the ex- HIF-1α or lncRNA BCRT1 knockdown attenuated the pression of lncRNA BCRT1 was negatively associated with effects induced by hypoxia, whereas lncRNA BCRT1 miR-1303, and a significant reciprocal repression feedback overexpression partly reversed the inhibitory effect of loop present in breast cancer cells. Importantly, miR-1303 RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 15 of 20 Fig. 7 LncRNA BCRT1 was transcriptionally regulated by HIF-1α during hypoxia. a-b The expression levels of HIF-1α protein (a) or lncRNA BCRT1 mRNA (b) in MDA-MB-231 and MDA-MB-468 cells were measured after culture under normoxia or hypoxia for 48 h by western blot or RT-PCR. c- d The efficiency of HIF-1α knockdown was detected using RT-PCR and western blot. e HIF-1α knockdown inhibited the expression of lncRNA BCRT1 in MDA-MB-231 and MDA-MB-468 cells under normoxia or hypoxia. f The recognition motif of HIF-1α from the JASPAR database. g Schematic illustration of the proximal region of the lncRNA BCRT1 promoter and the putative hypoxia responsive elements (HREs). h MDA-MB- 231 cells were transfected with a lncRNA BCRT1 promoter-containing pGL3 reporter vector and further treated with hypoxia or hypoxia combined with siHIF-1α. After 48 h, Luciferase activity was measured with the dual-luciferase reporter assay system. i ChIP assays with anti-HIF-1α antibody were performed to verify the binding between HIF-1α and the HREs of the lncRNA BCRT1 promoter under normoxia and hypoxia. j PTBP3 and HIF-1α expression from the TCGA breast cancer dataset was analyzed by the starBase database. k The mRNA and protein expression of PTBP3 was elevated under hypoxic conditions. l After HIF-1α knockdown, the expression of PTBP3 was evaluated by RT-PCR and western blot in MDA-MB-231 and MDA-MB-468 cells under normoxia or hypoxia. (*P < 0.05, **P < 0.01, and ***P < 0.001) RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 16 of 20 Fig. 8 LncRNA BCRT1 is essential for HIF-1α-mediated hypoxia-induced malignant properties. MDA-MB-231 cells were treated with normoxia, hypoxia, a combination of siHIF-1α and hypoxia, a combination of si-BCRT1 and hypoxia, or hypoxia and cotransfection with siHIF-1α and the pcDNA3.1-BCRT1 plasmid. a The expression of HIF-1α and PTBP3 was assessed by western blot. b The expression of lncRNA BCRT1 and PTBP3 was evaluated by RT-PCR. c Colony formation assay was used to evaluated the proliferation of MDA-MB-231 cells. d Monolayer morphology of MDA-MB-231 cells were photographed. e-f Transwell assay and wound healing assay were used to analyze the migration ability of MDA-MB-231 cells. (*P < 0.05, **P < 0.01, and ***P < 0.001) acted as a tumor suppressor in breast cancer, and miR- behavior of various cancers. PTBP3 was found to be up- 1303 overexpression partially reversed lncRNA BCRT1 regulated in gastric cancer compared with normal gastric overexpression-mediated promotion of proliferation, mi- mucosa [40], and high PTBP3 expression was correlated gration, invasion, and angiogenesis of breast cancer cells. with poor prognosis and higher lymph node metastasis Together, our results revealed that lncRNA BCRT1 could in gastric cancer patients. Further study revealed that serve as a ceRNA by sponging miR-1303 in breast cancer. PTBP3 was positively associated with metastasis of gas- Polypyrimidine tract-binding protein 3 (PTBP3), an tric cancer by regulating CAV1 through alternative spli- essential RNA-binding protein with roles in RNA alter- cing [25]. Moreover, a prooncogenic role for PTBP3 has native splicing (AS) [39], plays an important role in also been discovered in hepatocellular carcinoma medi- regulating gene expression and affects the biological ated by regulation of the splicing balance of NEAT1 and RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 17 of 20 pre-miR-612 [26]. In addition, previous studies reported 30–100 nm vesicles, can be secreted by cancer cells and in- that PTBP3 knockdown led to increased apoptosis and fluence tumor progression or drug resistance by modulat- cell cycle arrest, either through regulation of p53 signal- ing other cells in the microenvironment via intercellular ing [41] or through HDAC6-mediated inhibition of the communication [46]. Various exosomal lncRNAs have been phosphorylation of Akt and thymidylate synthase reported to participate in intercellular communication and (TYMS) expression [42]. However, the physiological are associated with the diagnosis and prognosis of cancer roles or molecular functions of PTBP3 in breast cancer [47]. Our results revealed that breast cancer cell-derived remain largely unclear, except one study that reported exosomes could promote M2 polarization and enhance its that PTBP3 promoted cell proliferation, migration, and tumor-promoting function by transmitting lncRNA invasion of breast cancer cells by preventing ZEB1 BCRT1. Nevertheless, the correlation between exosomal mRNA degradation [43]. However, the regulatory mech- lncRNA BCRT1 expression and the diagnostic or prognos- anism involved in the expression and function of PTBP3 tic values in breast cancer still needs further investigation. in breast cancer has not been fully elucidated. In the Hypoxia is a common phenomenon in various cancers current study, we identified PTBP3 as a target protein of and is associated with cancer progression. Several the lncRNA BCRT1/miR-1303 axis on the basis of the lncRNAs have been reported to be regulated by hypoxia following observations. Through bioinformatic predic- via HIF-1α-mediated transcriptional regulation. These tion and dual-luciferase reporter assays, PTBP3 was hypoxia-sensitive lncRNAs, such as lncRNA PVT1 [48], demonstrated to be a direct target gene of miR-1303 in lncRNA HITT [49], and LncRNA-MTA2TR [50], partici- breast cancer cells. Moreover, lncRNA BCRT1 overex- pate in tumorigenesis and tumor metastasis. In our study, pression led to increased expression of PTBP3, which we identified lncRNA BCRT1 as a hypoxia-sensitive could be partially reversed by miR-1303 overexpression, lncRNA. Using the JASPAR database, we predicted two indicating a lncRNA BRCT1/miR-1303/PTBP3 axis in potential HREs in the promoter of lncRNA BCRT1. More- breast cancer. We also revealed a significant positive re- over, the expression of lncRNA BCRT1 was increased lationship between the expression of lncRNA BCRT1 during hypoxia, which could be repressed by HIF-1α and PTBP3 in breast cancer cells. Furthermore, we re- knockdown. Furthermore, ChIP and dual-luciferase re- vealed that PTBP3 was increased in breast cancer tissues porter assays verified the regulatory effect of HIF-1α on and that PTBP3 knockdown clearly inhibited the prolif- lncRNA BCRT1 transcription in response to hypoxia. eration, migration and invasion of breast cancer cells. Notably, our results showed that hypoxia led to increased Hence, we further demonstrated the oncogenic role of expression of PTBP3 and lncRNA BCRT1 knockdown re- PTBP3 and provided evidence for the posttranscriptional pressed hypoxia-induced PTBP3, while lncRNA BCRT1 regulation of PTBP3 by a lncRNA in breast cancer. overexpression partially reversed the inhibition of PTBP3 Recently, considerable attention has been focused on the expression by HIF-1α knockdown. These results indicated significance of the tumor environment on tumor progres- a novel indirect pathway for hypoxia-induced PTBP3 ex- sion, a complex community that includes cancer cells, pression that was stimulated by increased lncRNA BCRT1 cancer-associated fibroblasts (CAFs), and immune inflam- levels. Moreover, our results revealed that lncRNA BCRT1 matory cells [44]. The interaction between cancer cells and knockdown could suppress hypoxia-induced proliferation TAMs, one of the most abundant immune cells in various and migration, whereas lncRNA BCRT1 overexpression solid cancers, was correlated with tumor progression, drug could rescue these effects, which was repressed by HIF-1α resistance, and poor prognosis in cancer patients [45]. knockdown under hypoxic conditions. Therefore, our Based on their biological properties, macrophages are study provided novel evidence supporting lncRNA as a generally categorized into two major phenotypes, pro- link between hypoxia and cancer progression. inflammatory (M1) and anti-inflammatory (M2) macro- phages. Many studies have demonstrated that TAMs are Conclusions considered M2-like macrophages that are closely associated In summary, we identified hypoxia-responsive lncRNA with cancer progression. Our results showed that lncRNA BCRT1 as a tumor-promoter in breast cancer, and the BCRT1 was increased in M2-like macrophages compared higher expression of lncRNA BCRT1 was associated with to M1-like macrophages and unpolarized macrophages. tumor metastasis and poor prognosis. LncRNA BCRT1 Moreover, lncRNA BCRT1 overexpression remarkably pro- acted as a sponge for miR-1303 to attenuate its repressive moted the expression of markers of M2-like macrophages, effect on PTBP3 and promoted M2 polarization through whereas lncRNA BCRT1 knockdown produced the oppos- exosome-mediated transfer. Our results provide a better ite results, indicating a promoting role of lncRNA BCRT1 understanding of the role of lncRNAs in breast cancer in M2 polarization. The conditioned medium of breast can- progression and a potential therapeutic target and prog- cer cells could influence the polarization of macrophages, nostic predictor against this malignancy. indicating the existence of a transfer mediator. Exosomes, RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 18 of 20 Supplementary information detect the expression of TGFβ in macrophages after indicated treatment. Supplementary information accompanies this paper at https://doi.org/10. (**P < 0.01, ***P < 0.001, Student’s t test). 1186/s12943-020-01206-5. Additional file 12: Figure S7. LncRNA BCRT1 is essential for HIF-1α- mediated hypoxia-induced malignant properties. MDA-MB-468 cells were Additional file 1: Table S1. Primers used for RT-PCR and vector treated with normoxia, hypoxia, a combination of siHIF-1α and hypoxia, a construction. combination of si-BCRT1 and hypoxia, or hypoxia further and cotransfec- Additional file 2: Table S2. Antibodies used in the experiments. tion with siHIF-1α and the pcDNA3.1-BCRT1 plasmid. a The expression of HIF-1α and PTBP3 was evaluated by western blot. b RT-PCR was used to Additional file 3: Figure S1. The sequence, secondary structure and detect the expression of lncRNA BCRT1 and PTBP3. c Proliferation of coding capacity of lncRNA BCRT1. a Schematic diagram showing the MDA-MB-468 cells was assessed by colony formation assay. d-e Transwell genomic locus of lncRNA BCRT1 in humans. Pink rectangles represent assay and wound healing assay were applied to analyze the migration exons. b The sequence of lncRNA BCRT1. c The secondary structure of ability of MDA-MB-468 cells. (*P < 0.05, **P < 0.01, and ***P < 0.001). lncRNA BCRT1 from AnnoLnc (http://annolnc.cbi.pku.edu.cn/). d Putative ORFs of lncRNA BCRT1 were predicted by the ORF Finder. e The amino acid sequences of the putative proteins. f The coding potential of lncRNA Abbreviations BCRT1 was measured by 5 different metrics and the results showed that LncRNAs: Long non-coding RNAs; GEO: Gene Expression Omnibus; lncRNA BCRT1 had no coding potential. TCGA: The Cancer Genome Atlas; ceRNA: competing endogenous RNA; NC: Negative control; EMT: Epithelial–mesenchymal transition; RIP assay: RNA Additional file 4: Table S3. Correlation between LncRNA BCRT1 immunoprecipitation assay; ChIP: Chromatin immunoprecipitation expression and clinicopathological features in breast cancer patients. Additional file 5: Table S4. Univariate analysis of overall survival in Acknowledgements breast cancer patients (n = 68). Not applicable. Additional file 6: Table S5. Multivariate analysis of overall survival in breast cancer patients (n = 68). Authors’ contributions Additional file 7: Figure S2. LncRNA BCRT1 regulates proliferation and YRL and QFY conceived the study; YRL, XJS, YML, HWZ, YL, and DWH migration of breast cancer cells in vitro. a The efficiency of lncRNA BCRT1 performed the experiments; BC, WJZ, NZ, TTM, LJW, and XYL collected knockdown in MCF-7 cells was validated with RT-PCR. b MTT assays clinical samples; YJW, FZY, and DL analyzed the data; YRL and XJS wrote the showed the reduced proliferation of MCF-7 cells transfected with si- paper; YRL and QFY revised the paper. All authors read and approved the BCRT1. c Colony formation assay showed the decreased proliferation of final manuscript. MDA-MB-231 and MDA-MB-468 cells after lncRNA BCRT1 knockdown. d The proliferation rate of MCF-7 cells was evaluated after lncRNA BCRT1 Funding overexpression. e-f MTT assay and colony formation assay were used to This work was supported by National Natural Science Foundation of China evaluate proliferation rate after lncRNA BCRT1 overexpression in MCF-7 (No.81272903; No.81672613; No.81874119; No. 81502285; No.81602329), cells. g-h Transwell assays demonstrated that lncRNA BCRT1 knockdown China Postdoctoral Science Foundation (No. 2018 M630787), Shandong inhibited whereas lncRNA BCRT1 overexpression promoted cell migration Science and Technology Development Plan (No. 2016CYJS01A02) and and invasion abilities in MCF-7 cells. (**P < 0.01, ***P < 0.001, Student’st Special Support Plan for National High-Level Talents (Ten Thousand Talents test). Program W01020103). Additional file 8: Figure S3. LncRNA BCRT1 and miR-1303 could mutu- Availability of data and materials ally regulate each other and miR-1303 overexpression inhibited cell prolif- The datasets used and/or analyzed during the current study are available eration and metastasis in vitro. a RT-PCR was used to validate the change from the corresponding author on reasonable request. of miR-1303 levels after lncRNA BCRT1 overexpression in MDA-MB-231 and MDA-MB-468 cells. b LncRNA BCRT1 expression was increased in Ethics approval and consent to participate tumor tissues from lncRNA BCRT1-overexpressing group compared to This project was approved by the Ethical Committee on Scientific Research control group. MiR-1303 expression in tumor tissues from lncRNA BCRT1- of Shandong University Qilu Hospital. overexpressing group was lower than those from control group. c Over- expression of miR-1303 was associated with better overall survival of Consent for publication breast cancer patients according to the LinkedOmics databases. d The ef- All human tissue samples were obtained with written informed consent ficiency of of miR-1303 overexpression in MCF7 cells was validated by RT- from all subjects. PCR. e MTT assays showed the reduced proliferation in miR-1303- overexpresing MCF-7 cells. f Transwell migration assays demonstrated Competing interests that miR-1303 overexpression inhibited cell migration. Columns are the The authors declare that they have no competing interests. average of three independent experiments. g LncRNA BCRT1 expression was decreased in MDA-MB-231 and MDA-MB-468 cells transfected with Received: 24 January 2020 Accepted: 23 April 2020 miR-1303 mimics. 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RETRACTED ARTICLE: LncRNA BCRT1 promotes breast cancer progression by targeting miR-1303/PTBP3 axis

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Springer Journals
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Copyright © The Author(s). 2020
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10.1186/s12943-020-01206-5
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Abstract

Background: Long noncoding RNAs (lncRNAs) play crucial roles in tumor progression and are aberrantly expressed in various cancers. However, the functional roles of lncRNAs in breast cancer remain largely unknown. Methods: Based on public databases and integrating bioinformatics analyses, the overexpression of lncRNA BCRT1 in breast cancer tissues was detected and further validated in a cohort of breast cancer tissues. The effects of lncRNA BCRT1 on proliferation, migration, invasion and macrophage polarization were determined by in vitro and in vivo experiments. Luciferase reporter assay and RNA immunoprecipitation (RIP) were carried out to reveal the interaction between lncRNA BCRT1, miR-1303, and PTBP3. Chromatin immunoprecipitation (ChIP) and RT-PCR were used to evaluate the regulatory effect of hypoxia-inducible factor-1α (HIF-1α) on lncRNA BCRT1. Results: LncRNA BCRT1 was significantly upregulated in breast cancer tissues, which was correlated with poor prognosis in breast cancer patients. LncRNA BCRT1 knockdown remarkably suppressed tumor growth and metastasis in vitro and in vivo. Mechanistically, lncRNA BCRT1 could competitively bind with miR-1303 to prevent the degradation of its target gene PTBP3, which acts as a tumor-promoter in breast cancer. LncRNA BCRT1 overexpression could promote M2 polarization of macrophages, mediated by exosomes, which further accelerated breast cancer progression. Furthermore, lncRNA BCRT1 was upregulated in response to hypoxia, which was attributed to the binding of HIF-1α to HREs in the lncRNA BCRT1 promoter. Conclusions: Collectively, these results reveal a novel HIF-1α/lncRNA BCRT1/miR-1303/PTBP3 pathway for breast cancer progression and suggest that lncRNA BCRT1 might be a potential biomarker and therapeutic target for breast cancer. Keywords: LncRNA BCRT1, miR-1303, PTBP3, Progression, Breast cancer * Correspondence: qifengy_sdu@163.com Department of Breast Surgery, Qilu Hospital of Shandong University, Jinan, Shandong 250012, People’s Republic of China Pathology Tissue Bank, Qilu Hospital of Shandong University, Jinan, Shandong 250012, People’s Republic of China © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 2 of 20 Background In the present study, we analyzed public microarrays Breast cancer is one of the most common malignancies to screen lncRNAs that are differentially expressed in among women worldwide. Despite advances in diagnosis breast cancer. LncRNA BCRT1 (breast cancer-related and combined treatments, the prognosis of breast cancer transcript 1), which was significantly overexpressed in patients remains unsatisfactory [1, 2]. Metastasis is one breast cancer tissues and associated with poor prognosis of the leading causes of cancer-related death [3], which of breast cancer patients, was selected for further investi- greatly hinders treatment success. Therefore, a more gation. LncRNA BCRT1 functioned as a tumor promoter comprehensive understanding of the mechanism of pro- by competitively binding with miR-1303 to protect PTBP3 gression and metastasis is important for improving the from degradation and thus promoted the growth and pro- prognosis of breast cancer patients. gression of breast cancer cells both in vitro and in vivo. Recently, long noncoding RNAs (lncRNAs) have been Moreover, lncRNA BCRT1 could be transferred to macro- found to be involved in a variety of physiological and phages via exosomes, promoting M2 polarization and en- pathological processes [4, 5], especially in cancers [6]. hancing its effect on tumor progression. Further study LncRNAs are transcripts with more than 200 nucleotides revealed that lncRNA BCRT1 was induced by hypoxia via that have no protein-coding potential [7]. Despite the HIF-1α-dependent transcriptional regulation, which conse- lack of cross-species conservation [8], researchers in our quently facilitated hypoxia-induced EMT. Our results pro- laboratory and others have demonstrated that lncRNAs vide novel insight into the metastatic mechanism of breast are frequently dysregulated in cancers and are involved cancer and a promising therapeutic target for breast cancer in the progression and metastasis of multiple malignan- treatment. cies [9, 10]. LncRNA ANCR was found to mediate the degradation of EZH2 and thus attenuate the metastatic Methods ability of breast cancer [11]. Moreover, lncRNA AGAP2- Patients and specimens AS1 was found to be upregulated in breast cancer and Human breast cancer tissues and corresponding normal tis- was associated with trastuzumab resistance [12]. How- sues were obtained from patients admitted to Qilu Hospital ever, the clinical significance and biological mechanisms from January 2004 to December 2011. All participants of the vast majority of lncRNAs in the regulation of provided written informed consent, and the research was breast cancer remain largely unknown. approved by the Ethical Committee on Scientific Research Several studies have suggested that lncRNAs may func- of Shandong University Qilu Hospital. tion as competing endogenous RNAs (ceRNAs) to regulate the biological functions or expression of microRNAs. For instance, lncRNA LINC00963 promotes tumorigenesis and RNA sequencing analysis radioresistance by acting as a ceRNA for miR-324-3p in Breast cancer gene expression data were downloaded breast cancer cells [13]. LncRNA NONHSAT101069 acted from The Cancer Genome Atlas (TCGA) and the Gene as a ceRNA by effectively sponging miR-129-5p, thereby Expression Omnibus (GEO) dataset GSE112848. The modulating the repression of Twist1 and promoting epiru- data analysis was performed with R software using the bicin resistance, migration, and invasion of breast cancer DEGseq package. The threshold set for significant differ- cells [14]. Previous studies have revealed that hypoxia, a ences was log |fold change| ≥ 1 and P-value < 0.05. major hallmark of the tumor microenvironment, is associ- ated with the progression and metastasis of many solid tu- mors. HIF-1α is an extensively studied hypoxia-inducible Cell culture and reagents factor (HIF) that mediates the cellular response to hypoxia All cell lines were purchased from the American Type through transactivation of downstream target genes [15]. Culture Collection (Manassas, VA) and were cultured ac- Under normoxic conditions, HIF-1α is subjected to prote- cording to the manufacturer’s instructions. MCF10A cells asome degradation, whereas, hypoxic conditions protect were cultured in Dulbecco’smodifiedEagle’smedium HIF-1α from degradation, allowing HIF-1α translocation (Invitrogen, USA) containing 5% horse serum, 10 μg/ml into the nucleus to initiate gene expression [16]. Re- insulin, 20 ng/ml EGF, 100 ng/ml cholera toxin, and cently, the roles of hypoxic conditions in regulating 0.5 μg/ml hydrocortisone. MCF-7, MDA-MB-231, MDA- lncRNA expression have received extensive attention, MB-468, and HEK293T cells were cultured with Dulbec- and various hypoxia-responsive lncRNAs have been co’smodifiedEagle’s medium. T47D and THP1 cells were reported to play important roles in tumorigenesis and cultured with RPMI 1640 medium. The above media con- tumor progression [17]. However, more investigations tained 100 U/ml penicillin, 100 μg/ml streptomycin and should be carried out on the mechanism of hypoxia 10% fetal bovine serum (Invitrogen, USA). The medium in mediating aberrant lncRNA expression as well as for T47D cells also contained 10 μg/ml insulin. All cells the functions of lncRNAs in breast cancer. were cultured in a 5% CO2-humidified incubator at 37 °C. RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 3 of 20 RNA extraction and quantitative real-time PCR analysis curves were determined by calculating the relative value Total RNA was isolated using TRIzol reagent (Invitrogen, of absorbance measured at 570 nm on a microplate USA). Complementary DNA (cDNA) was synthesized reader (Bio-Rad, USA). using a PrimeScript RT reagent kit (TaKaRa, Japan). For miRNAs, reverse transcription was carried out using the Colony formation assay PrimeScript miRNA cDNA Synthesis Kit (TaKaRa, Japan). Transfected cells were counted and seeded at 500 cells RT-PCR was performed using SYBR Premix Ex Taq I. per 6 cm plate. After 10–14 days, cell colonies were Primers used in the study are listed in Additional file 1: washed with PBS, fixed with ethanol for 15 min and Table S1. β-Actin was used as an internal control for stained with crystal violet for 20 min. The colonies were mRNA. U6 was used as an internal control for miRNA. imaged and counted. Relative RNA abundances were calculated by the standard -ΔΔCt 2 method. EdU incorporation assay Transfected cells were seeded into 96-well plates at a Subcellular fractionation density of 1 × 10 cells/well. The EdU incorporation Nuclear and cytoplasmic separation was performed assay kit (RiboBio, China) was used to evaluate cell pro- using the PARIS Kit (Life Technologies, USA) according liferation. A fluorescence microscope (Nikon, Japan) was to the manufacturer’s instructions. used to obtain images. Fluorescence in situ hybridization (FISH) Cell apoptosis assay The FISH assay was performed in MDA-MB-231 cells ac- EDTA-free trypsin was used to collect cells, and the cells cording to the specifications of the manufacturers. The were resuspended in 500 μl of binding buffer. After incu- Cy3-labeled lncRNA BCRT1 probes used in our study bation with 5 μl Annexin V-FITC and 5 μl PI (BD Biosci- were designed and synthesized by GenePharma (Shanghai, ences, USA) for 15 min in the dark, the cells were China). Briefly, the prepared cells were fixed with 4% para- examined on a FACSCalibur (BD, Biosciences, USA) formaldehyde for 30 min. After permeabilization, the cells within 1 h. were incubated with specific probes at 37 °C overnight. The cell nuclei were stained with DAPI (Sigma-Aldrich, Transwell assay USA). The staining results were observed using a fluores- Transwell assays were performed using Transwell cham- cence microscope (Nikon, Japan). bers (pore size 8 μm; Costar Corporation, USA) with or without matrigel (BD Biosciences, USA). A total of 1 × Plasmid construction and transfection 10 cells were added to the upper insert. The lower The full-length lncRNA BCRT1 cDNA was cloned into chamber contained 700 μl medium with 20% FBS as a pcDNA3.1 (Invitrogen, USA). The primers used for vector chemoattractant. After incubation for 24–48 h, the cells construction are showed in Additional file 1:Table S1.The on the lower surface were fixed with ethanol and stained lncRNA BCRT1 plasmid and corresponding empty vector with 0.2% crystal violet. The relative cell number was were transfected into breast cancer cells using Lipofecta- calculated. mine 2000 reagent (Invitrogen, USA). G418 (2 mg/ml) was used to generate stably transfected cells. For PTBP3 knock- Tube formation assay down, the pLKO.1 plasmid was used as a negative control. Seventy-five microliters of Matrigel (BD Biosciences, The 3′UTR sequences of lncRNA BCRT1 and PTBP3 with USA) was pipetted into each well of a 48-well plate and wild-type or mutant miR-1303 binding sites were cloned allowed to solidify for more than 1 h at 37 °C. HUVECs into the pmirGLO vector (Invitrogen, USA). Different frag- were suspended in the indicated conditioned medium ments of the lncRNA BCRT1 promoter were cloned into and seeded onto the gel. After 4–6 h of incubation, a the pmirGLO vector. The negative control, lncRNA BCRT1 bright-field microscope was used to observe the tubular siRNAs, and miR-1303 mimics (GenePharma, China) were structures and acquire images. Tube formation was transfected using Lipofectamine 2000. quantified by measuring the total length of the tubes using ImageJ software. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay Western blot assay Transfected cells were seeded at a density of 5000 cells/ Cell proteins were extracted and separated by 10% SDS- well in 96-well plates. After incubation, 20 μl of 5 mg/ml PAGE gels and transferred to 0.22 μm PVDF membranes MTT was added to each well and incubated for another (Millipore, USA). The membranes were blocked with 5% 4 h. Then, the supernatants were carefully removed, and skim milk powder and incubated with specific antibodies 100 μl DMSO was added to each well. The proliferation at 4 °C overnight. The membranes were then incubated RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 4 of 20 with the appropriate secondary antibodies, and an ECL Antibodies used for the RIP assay included anti-AGO2 detection system (Bio-Rad, USA) was used to detect the and control IgG (Millipore, USA), and the coprecipitated protein bands. β-Actin was used as a control. The primary RNAs were used for cDNA synthesis and evaluated by antibodies and secondary antibodies used are described in qRT-PCR. Additional file 2: Table S2. Isolation and characterization of exosomes Tumor xenograft model Exosomes were isolated from the supernatant of MDA- MDA-MB-231 cells (1 × 10 cells) with or without MB-231 cells that had been cultured in DMEM containing lncRNA BCRT1 overexpression were suspended in 10% exosome-depleted FBS for 48 h via a polyethylene 200 μl PBS and subcutaneously injected into each flank glycol-based method as previously described [18]. Briefly, of 4–6-week-old BALB/c nu/nu female mice. The mice the culture medium was centrifuged at 500×g for 5 min, were sacrificed after 30 days, and the maximum (L) and and the supernatant was further centrifuged at 2000×g for minimum (W) length and weight of the tumors were 30 min. Then, 2× PEG solution was added to the super- measured. Tumor volume was calculated as ½LW .To natant and gently mixed. The mixture was stored at 4 °C evaluate the influence of lncRNA BCRT1 on metastasis, formore than12h andthen centrifuged at 10,000×g for 1 5×10 cells were injected into the lateral tail veins of h at 4 °C to collect the exosomes. The supernatant was re- nude female mice (five mice per group). After 4 weeks, moved, and the exosome pellet was resuspended in 0.2 μm- the mice were euthanized, and the lungs were collected filtered PBS. to evaluate the number of pulmonary metastatic lesions. Hematoxylin and eosin (H&E) staining was performed Exosome uptake assay for tissue morphology evaluation. The animal experi- PKH26, a red fluorescent dye (Sigma-Aldrich, USA), was ments were approved by the Shandong University Ani- used to label exosomes obtained from conditioned mal Care and Use Committee. medium [19]. After incubation with the recipient cells for 12 h, fluorescence microscopy was used for imaging. Immunohistochemical (IHC) analysis The paraffin-embedded sections were dewaxed in xylene Kaplan-Meier plotter tool analysis and rehydrated in alcohol. Endogenous peroxidase was The Kaplan-Meier Plotter tool (http://kmplot.com/analysis/) blocked by 3% H O , and microwave heating was per- 2 2 was used to determine the association between PTBP3 and formed for antigen retrieval. After blocking nonspecific the prognosis of breast cancer patients. antigen binding with 5% BSA at 37 °C for 1 h, the sections were incubated with a specific primary antibody against Chick chorioallantoic membranes (CAM) Ki67, PTBP3 or CD31 (1100 dilution, Abcam, USA) at The fertilized chicken eggs were cultured at 37 °C in an 4 °C overnight. After incubating with the corresponding 80% humidified atmosphere for 7 days. Then, a square secondary antibodies at 37 °C for 1 h, the sections were window was cut on the shell to expose the CAM and was stained with diaminobenzidine and counterstained with covered with a gelatin sponge (0.3 cm × 0.3 cm × 0.3 cm) hematoxylin. Representative images were taken using an containing PBS or the indicated conditioned medium Olympus light microscope. (CM). Next, tape was used to cover the window for further incubation. After 2 days, the CAM were visualized under Luciferase assay astereoscope. The wild-type or mutant lncRNA BCRT1 or 3’UTR of PTBP3 was amplified and cloned into pmirGLO vector Chromatin immunoprecipitation (ChIP) separately. Then, HEK293T cells were plated on a 96- ChIP assays were performed using a ChIP kit (CST, well plate and cotransfected with wild-type or mutant USA) following the manufacturer’s instructions. Briefly, luciferase plasmids and miR-1303 or control miRNA. cells were crosslinked with formaldehyde and sonicated The lncRNA BCRT1 promoter segment was cloned into to an average length of 200–1000 bp. Immunoprecipita- the pGL3-basic vector. The pGL3-BCRT1 and pRL-TK tion was conducted with an anti-HIF-1α antibody vectors were cotransfected with si-NC or si-HIF1α.A (Abcam, UK) or IgG control. Precipitated DNA was Dual-Luciferase Reporter Assay System (Promega, USA) amplified by RT-PCR. Primer sequences are provided in was used to measure the luciferase activity. Supplementary Table S4. RNA immunoprecipitation (RIP) assay A Magna RIP RNA-Binding Protein Immunoprecipita- Elisa tion Kit (Millipore, USA) was used to determine the re- The TGFβ concentration in the cell culture medium was lationship between lncRNA BCRT1 and miR-1303. measured by ELISA using the Quantikine human TGFβ RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 5 of 20 ELISA kit (R&D Systems, USA) according to the manu- expression levels were correlated with significantly facturer’s instructions. shorter disease-free survival (DFS) and overall survival (OS) (Fig. 1e). Univariate (Additional file 5: Table S4) Statistical analysis and multivariate (Additional file 6: Table S5) analyses Data are expressed as the mean ± S.D. of three inde- further showed that lncRNA BCRT1 expression was a pendent experiments and analyzed by the SPSS software major prognostic factor for breast cancer patients. The program (version 17.0). Student’s t-test was used for results of nuclear/cytoplasmic RNA fractionation from two-group comparisons. Kaplan–Meier survival analysis the subcellular distribution assay confirmed that lncRNA was performed for survival rate calculation. Cox propor- BCRT1 was mainly located in the cytoplasm (Fig. 1f), tional hazards model multivariate analyses were used to which was further confirmed by the fluorescence in situ evaluate the significance of lncRNA BCRT1 expression hybridization (FISH) analysis (Fig. 1g). Collectively, these and clinicopathological features on overall survival. P < findings revealed that lncRNA BCRT1 was upregulated 0.05 was considered statistically significant. in breast cancer and that high expression of lncRNA BCRT1 was associated with poor outcomes in breast Results cancer. LncRNA BCRT1 expression is upregulated in breast cancer and associated with poor prognosis LncRNA BCRT1 promotes cell proliferation and tumor To identify important lncRNAs that potentially participate growth in breast cancer in breast cancer progression, we analyzed the lncRNA ex- To determine the biological function of lncRNA BCRT1 in pression profiles using public databases (GSE112848 and a breast cancer cells, short interference siRNAs against hu- TCGA dataset) (Fig. 1a-b). In the present study, we mainly man lncRNA BCRT1 (si-BCRT1) were applied to knock focused on the upregulated lncRNAs given that these down lncRNA BCRT1, and the knockdown efficiency was lncRNAs might serve as therapeutic targets or prognostic confirmed by RT-PCR (Fig. 2a and Additional file 7:Figure biomarkers. Among them, lncRNA BCRT1 (breast cancer S2a). After lncRNA BCRT1 knockdown, the proliferation, related transcript 1), which was one of the prominently colony-formation abilities, and DNA synthesis activities of upregulated lncRNAs in breast cancer tissues, was chosen breast cancer cells were significantly decreased (Fig. 2b-d for further evaluation. LncRNA BCRT1 is located on and Additional file 7: Figure S2b). The results of flow cy- 10q25.1 in humans and is composed of 3 exons with a full tometry revealed that lncRNA BCRT1 knockdown obvi- length of 1013 nt (Additional file 3:Figure S1a). These- ously increased the total apoptosis rate in breast cancer quence of full-length lncRNA BCRT1 and its secondary cells (Fig. 2e). On the other hand, when lncRNA BCRT1 structure based on minimum free energy (MFE) are was overexpressed by transfection with the pcDNA3.1 plas- shown in Additional file 3: Figure S1b and c, respectively. mid containing the lncRNA BCRT1 sequence, the prolifera- Moreover, using the Open Reading Frame (ORF) Finder tion and colony formation of breast cancer cells was and conserved domain database, we found that lncRNA significantly increased (Fig. 2f-g and Additional file 7: BCRT1 had little potential to code proteins, which was in Figure S2c-f). Furthermore, a subcutaneous xenograft accordance with the results of five different online metrics model was used to validate the biological function of (Additional file 3: Figure S1d-f). In addition, we failed to lncRNA BCRT1 in vivo. Consistent with the results identify a valid Kozak consensus sequence in lncRNA in vitro, lncRNA BCRT1 overexpression significantly in- BCRT1, further supporting the notion that lncRNA creased tumor weight and tumor volume compared with BCRT1 had no protein-coding potential [20]. those in the control group (Fig. 2h-i). Moreover, immuno- Compared with that in normal breast epithelial cells histochemistry (IHC) assays confirmed that lncRNA (MCF10A), the expression of lncRNA BCRT1 in four BCRT1 overexpression caused increased Ki67 expression breast cancer cell lines was significantly higher (Fig. 1c). (Fig. 2j), indicating enhanced cell proliferation. Our findings Moreover, we further investigated the lncRNA BCRT1 indicated that lncRNA BCRT1 could promote breast cancer expression levels in 18 paired breast cancer tissues and cell proliferation both in vitro and in vivo. normal breast tissues using real-time PCR analysis, and the results revealed that lncRNA BCRT1 was signifi- LncRNA BCRT1 promotes cell mobility and tumor cantly overexpressed in breast cancer tissues compared metastasis in breast cancer with adjacent normal tissues (Fig. 1d). The association We then investigated the role of lncRNA BCRT1 in the between the clinicopathological characteristics of breast motility of breast cancer cells. The results showed that cancer patients and lncRNA BCRT1 expression level is lncRNA BCRT1 knockdown significantly impaired the summarized in Additional file 4: Table S3. LncRNA migration and invasion of breast cancer cells, whereas BCRT1 was overexpressed in breast cancer tissues with lncRNA BCRT1 overexpression led to increased cell mo- distant metastasis (Fig. 1d), and higher lncRNA BCRT1 bility (Fig. 3a-b and Additional file 7: Figure S2g-h). RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 6 of 20 Fig. 1 LncRNA BCRT1 upregulation is associated with advanced progression and poor prognosis in breast cancer. a Heat maps showing the top differentially expressed lncRNAs in breast cancer samples compared to normal tissues (left, GSE112848; right, TCGA). The red shades represent high expression, and green shades represent low expression. b Volcano plots showing the expression profiles of lncRNAs. c-d RT-PCR analysis was used to detect the expression of lncRNA BCRT1 in cell lines and tissues. Actin was the internal control. e Kaplan–Meier analysis showed the association between lncRNA BCRT1 expression and disease-free survival or overall survival of breast cancer patients (n = 68). f The expression level of lncRNA BCRT1 in the subcellular fractions of MDA-MB-231 cells was detected by qRT-PCR. U6 and GAPDH were used as nuclear and cytoplasmic markers, respectively. g The location of lncRNA BCRT1 (red) in MDA-MB-231 cells was determined by FISH assay. DAPI-stained nuclei are blue. (**P < 0.01 and ***P < 0.001) Moreover, we used breast cancer-conditioned medium to length (Fig. 3c). Given that epithelial-mesenchymal transi- stimulate angiogenesis in HUVECs to evaluate angiogenesis tion (EMT) is one of the major mechanisms for cancer me- activity in vitro [21]. The results showed that the relative tastasis, we further evaluated the effect of lncRNA BCRT1 length of tubes was decreased in the si-BCRT1 group com- on EMT-related markers. Western blot analysis showed pared with the si-NC group. On the other hand, lncRNA that lncRNA BCRT1 knockdown could increase the expres- BCRT1overexpressionled to asignificantlyelevatedtube sion of epithelial markers (E-cadherin) and decrease the RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 7 of 20 Fig. 2 LncRNA BCRT1 knockdown inhibited breast cancer cell proliferation in vitro and in vivo. a The expression levels of lncRNA BCRT1 in MDA- MB-231 and MDA-MB-468 cells after transfection with si-NC or si-BCRT1 were detected by RT-PCR. b-c The effects of lncRNA BCRT1 knockdown on the proliferation of MDA-MB-231 and MDA-MB-468 cells were examined by MTT assay (b) and colony formation assays (c). Experiments were performed in triplicate. d EdU assays were used to detect the proliferation rate of MDA-MB-231 and MDA-MB-468 cells after lncRNA BCRT1 knockdown. Columns are the average of three independent experiments. e Flow cytometry was performed to determine the effect of lncRNA BCRT1 on apoptosis by flow cytometry analysis. f RT-PCR was used to determine the efficiency of the lncRNA BCRT1-overexpressing vector. g MTT assay indicated an increased proliferative ability of MDA-MB-231 and MDA-MB-468 cells after lncRNA BCRT1 overexpression. h MDA-MB-231 cells were stably transfected with the lncRNA BCRT1-overexpressing vector or control vector and injected subcutaneously into nude mice. Compared with the vector group, lncRNA BCRT1 overexpression promoted tumor growth. i Tumor volume and weight were significantly increased in the lncRNA BCRT1-overexpressing group. j Representative images of H&E and Ki67 staining in the tumor. Immunohistochemical staining revealed that lncRNA BCRT1 overexpression led to increased expression of Ki67. (**P < 0.01 and ***P < 0.001) RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 8 of 20 Fig. 3 (See legend on next page.) RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 9 of 20 (See figure on previous page.) Fig. 3 LncRNA BCRT1 knockdown inhibited breast cancer cell metastasis in vitro and in vivo. a Transwell migration and invasion assays were used to evaluate the motility of MDA-MB-231 and MDA-MB-468 cells transfected with si-NC or si-BCRT1. Columns are the average of three independent experiments. b LncRNA BCRT1 overexpression led to increased migration and invasion of MDA-MB-231 and MDA-MB-468 cells. c Tube formation in HUVECs was inhibited by conditioned medium from MDA-MB-231 cells transfected with si-BCRT1 and was promoted by that from MDA-MB-231 cells transfected with the lncRNA BCRT1-overexpressing vector. d EMT-related proteins were detected by western blot in MDA-MB-231 and MDA-MB-468 cells after knockdown or overexpression of lncRNA BCRT1. e Stably transfected MDA-MB-231 cells were injected into the tail veins of nude mice (n = 5). Representative images of lungs and H&E staining of lungs isolated from mice. LncRNA BCRT1 overexpression resulted in an increased number of lung metastatic colonies. f Representative immunohistochemistry staining of CD31 in the indicated xenografts. The corresponding statistical plots are presented in the lower panel. (**P < 0.01 and ***P < 0.001) expression of mesenchymal markers (such as Fibronec- breast cancer. A negative association between lncRNA tin, N-cadherin, and Vimentin) (Fig. 3d), indicating that BCRT1 and miR-1303 was also detected in xenograft tu- lncRNA BCRT1 could regulate the EMT process to mors (Additional file 8:FigureS3b). modulate breast cancer progression. To confirm these Then, we examined the role of miR-1303 in breast can- findings in vivo, we injected breast cancer cells through cer. Higher expression of miR-1303 was correlated with the tail vein to establish a pulmonary metastasis model better overall survival of breast cancer patients according to in nude mice. Two of the five mice (2/5) injected with the LinkedOmics database [24](Additional file 8:Figure breast cancer cells in the control group and all five S3c), indicating that miR-1303 acted as a tumor suppressor mice (5/5) injected with breast cancer cells in the in breast cancer. The transfection efficiency of miR-1303 lncRNA BCRT1-overexpressing group showed meta- mimics was determined by RT-PCR (Fig. 4eand Additional static foci in their lungs after 4 weeks (Fig. 3e). Then, file 8: Figure S3d), and miR-1303 overexpression led to a all mice were sacrificed, and their lungs were subjected decreased proliferation rate and increased apoptotic rate of to hematoxylin and eosin (H&E) staining. The results breast cancer cells (Fig. 4f-g, and Additional file 8:Figure revealed that lncRNA BCRT1 overexpression remark- S3e). Moreover, miR-1303 overexpression decreased cell ably increased the volume and number of lung meta- migration and invasion (Fig. 4h and Additional file 8:Figure static lesions compared with those in the control group S3f).Importantly,rescueexperiments further validated the (Fig. 3f). Similarly, vascular density was increased in the functional relationship between lncRNA BCRT1 and miR- lncRNA BCRT1-overexpressing group (Fig. 3f). Taken 1303 (Fig. 3i-k). Moreover, lncRNA BCRT1 expression was together, these data show that lncRNA BCRT1 pro- decreased after miR-1303 overexpression in breast cancer motes tumor metastasis in breast cancer cells. cells (Additional file 8: Figure S3g), indicating a reciprocal suppression between them. Overall, we chose miR-1303 as LncRNA BCRT1 functions as a miR-1303 sponge in breast an inhibitory target of lncRNA BCRT1 for further investiga- cancer cells tion in breast cancer. Recently, many lncRNAs have been reported to function as competing endogenous RNAs (ceRNAs) in modulating the LncRNA BCRT1 upregulates PTBP3 expression via expression and biological functions of miRNAs [22, 23]. inhibition of miR-1303 Since lncRNA BCRT1 was distributed predominantly in Using the miRDB, miRWalk, miRPathDB, and TargetS- the cell cytoplasm, we hypothesized that lncRNA BCRT1 can databases, we found that PTBP3 was a potential tar- might act as a miRNA sponge to prevent miRNAs from get of miR-1303 (Fig. 5a). Additionally, we found that binding with their target mRNAs. Through the RegRNA the expression of PTBP3 was elevated in breast cancer database, we identified miR-1303 as a potential target of tissues compared to normal tissues using the TCGA and lncRNA BCRT1 (Fig. 4a). To validate the binding potential, GEO databases (Fig. 5b), and high PTBP3 expression a luciferase reporter assay was performed. Overexpression was associated with poor prognosis of breast cancer pa- of miR-1303 significantly reduced the luciferase activity of tients (Additional file 9: Figure S4). Furthermore, we the pmirGLO-BCRT1-wt vector but failed to decrease that found that the expression of PTBP3 was positively asso- of the mutant vector (Fig. 4b). The AGO2 immunoprecipi- ciated with the expression of lncRNA BCRT1 in breast tation assay showed that the AGO2 antibody was able to cancer cells (Fig. 5c). Therefore, PTBP3 was selected as pull down both endogenous lncRNA BCRT1 and miR- a putative target of miR-1303 for further observation. 1303 (Fig. 4c), further validating their binding potential. Luciferase assays showed that overexpression of miR- Moreover, lncRNA BCRT1 knockdown promoted miR- 1303 decreased the luciferase activity of the wild-type 1303 expression (Fig. 4d), whereas lncRNA BCRT1 overex- PTBP3 reporter but not the mutant reporter (Fig. 5d), pression inhibited miR-1303 expression (Additional file 8: indicating that PTBP3 was the direct target of miR-1303. Figure S3a). Our above data supported the hypothesis that Furthermore, the mRNA and protein levels of PTBP3 miR-1303 is an inhibitory target of lncRNA BCRT1 in were reduced by miR-1303 overexpression (Fig. 5e) or RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 10 of 20 Fig. 4 LncRNA BCRT1 acts as a sponge of miR-1303 in breast cancer. a Schematic diagram representing the predicted binding sites for miR-1303 in lncRNA BCRT1 and mutant sequences of the potential miR-1303 binding sites. b Luciferase assays in HEK293T cells cotransfected with wild-type or mutant lncRNA BCRT1 and miR-1303 or NC. The data are shown as the means ± SD of triplicate samples. c Anti-AGO2 RIP was performed in HEK293T cells, followed by RT-PCR to detect the expression of lncRNA BCRT1 or miR-1303 associated with AGO2. d RT-PCR was used to detect the effect of lncRNA BCRT1 knockdown on the expression of miR-1303 in breast cancer cells. e The overexpression of miR-1303 in breast cancer cells was validated by RT-PCR. f The proliferation of breast cancer cells transfected with NC or miR-1303 was measured by MTT assay. g MDA-MB- 231 and MDA-MB-468 cells were transfected with miR-1303 mimics or NC, and the apoptotic rates were determined by FACS analysis. Representative results are shown, and data are presented as the mean ± SD. h Transwell assays were used to measure the migration of breast cancer cells transfected with miR-1303 mimics or NC. i The effects of lncRNA BCRT1 and miR-1303 cotransfection on cell proliferation were measured by MTT assay. j Transwell assay was used to determine the migration of breast cancer cells cotransfected with lncRNA BCRT1 and miR- 1303. k Overexpression of miR-1303 inhibited the effect of conditioned medium from lncRNA BCRT1-overexpressing cells on the tube formation of HUVECs. (*P < 0.05, **P < 0.01, and ***P < 0.001) RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 11 of 20 Fig. 5 LncRNA BCRT1 promoted breast cancer cell proliferation and progression by protecting PTBP3 from miR-1303-induced degradation. a Schematic illustration showing the overlapping target genes of miR-1303 predicted by miRDB, miRWalk, miRPathDB, and TargetScan. b The expression of PTBP3 was increased in breast cancer tissues compared to normal tissues based on the TCGA and GEO databases. c RT-PCR revealed a positive correlation between lncRNA BCRT1 expression and PTBP3 expression in breast cancer cells. d The upper schematic diagram represents the construction of the luciferase reporter plasmids. The lower panel shows the predicted and the mutated binding sites of miR-1303 in the 3′UTR of PTBP3. The statistical graphs on the right show the luciferase activity in HEK293T cells with or without miR-1303 overexpression and transfected with the WT or MUT luciferase plasmids. e RT-PCR and western blot assays revealed the effect of miR-1303 on PTBP3 expression. f RT-PCR and western blot assays showed that lncRNA BCRT1 knockdown repressed the expression of PTBP3. g RT-PCR and western blot assays were used to determine the PTBP3 expression level in MDA-MB-231 cells cotransfected with pcDNA3.1-BCRT1 and miR-1303 mimics. h RT-PCR was used to detect the efficiency of PTBP3 knockdown in breast cancer cells. i MTT assay was performed to examine the proliferation ability after PTBP3 knockdown. j PTBP3 knockdown led to increased cell apoptosis. k Transwell assays revealed that PTBP3 knockdown inhibited the migration and invasion abilities of breast cancer cells. (*P < 0.05, **P < 0.01, and ***P < 0.001) RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 12 of 20 lncRNA BCRT1 knockdown (Fig. 5f). In the rescue ex- attempted to investigate the mechanism mediating the periments, overexpression of miR-1303 could partly communication between breast cancer cells and macro- counteract the corresponding increases in PTBP3 ex- phages. Various studies have reported that lncRNAs can be pression induced by lncRNA BCRT1 overexpression in transferred by exosomes to modulate the tumor micro- breast cancer cells (Fig. 5g). In addition, lncRNA BCRT1 environment [31]. To investigate whether lncRNA BCRT1 overexpression also led to increased expression of can be packed into exosomes, we extracted exosomes from PTBP3 in xenograft tumors (Additional file 10: Figure the cultured supernatants of breast cancer cells and used S5a-b). Previous studies reported that PTBP3 acted as a western blotting to detect the expression of exosome- tumor promoter in various cancers, such as gastric can- related proteins, such as CD63, HSP70, and HSP90 (Fig. cer [25], hepatocellular carcinoma [26], and colorectal 6f). LncRNA BCRT1 overexpression in MDA-MB-231 cells cancer [27]. However, the role of PTBP3 in breast cancer led to increased levels of lncRNA BCRT1 in the secreted has not been fully elucidated. PTBP3 knockdown re- exosomes, whereas lncRNA BCRT1 knockdown produced sulted in significantly inhibited cell proliferation and in- the opposite results (Fig. 6g), indicating the existence of creased cell apoptosis (Fig. 5h-j). Moreover, Transwell lncRNA BCRT1 in exosomes. We labeled MDA-MB-231 assays showed that PTBP3 knockdown led to attenuated cell-derived exosomes with PKH26 and incubated them migration and invasion of breast cancer cells (Fig. 5k). with macrophages to examine exosome incorporation and These data suggested that PTBP3 acted as a tumor pro- confirmed that the labeled exosomal RNAs could be inter- moter in breast cancer, and lncRNA BCRT1 played signifi- nalized by macrophages (Fig. 6h). Then, we cocultured cant roles in regulating PTBP3 expression by regulating unpolarized macrophages with exosomes isolated from miR-1303. lncRNA BCRT1-overexpressing or control MDA-MB-231 cells. The expression of lncRNA BCRT1 and M2 pheno- Exosomal lncRNA BCRT1 promotes M2 phenotype type markers (CD206 and MRC-2) was significantly in- polarization and enhances macrophage-induced tumor creased in the lncRNA BCRT1-overexpressing group progression compared to the control group (Fig. 6i), indicating that Previous studies have reported that tumor-associated mac- exosomal lncRNA BCRT1 promoted M2 polarization. rophages (TAMs), which are considered to have an M2- Then, we investigated the role of lncRNA BCRT1 in like phenotype, are the most abundant immune-related modulating the behaviors of macrophages. As expected, cells in the tumor microenvironment (TME) and partici- supernatants from lncRNA BCRT1-overexpressing cells pate in tumor development by mediating angiogenesis, me- led to increased migration ability of macrophages and tastasis, and immune escape [28–30]. To investigate showed enhanced chemotaxis (Additional file 11: Figure whether lncRNA BCRT1 contributes to M2 polarization, S6a-b). Moreover, supernatant or exosomes from we evaluated the expression of lncRNA BCRT1, M1 lncRNA BCRT1-overexpressing cells promoted the markers, and M2 markers in unpolarized macrophages, expression and secretion of TGF-β compared with the LPS/INF-γ-induced M1 macrophages, and IL-4/IL-13-in- control groups (Additional file 11:FigureS6c-e). To duced M2 macrophages. The results revealed that the ex- further investigate whether lncRNA BCRT1-educated pression levels of M1-associated genes (CD80, MCP-1, M2 phenotype macrophages have the characteristic iNOS, and IL-6) were significantly upregulated in M1 mac- function of tumor promotion, we treated macrophages rophages, whereas those of M2-associated genes, including with exosomes or supernatants isolated from lncRNA CD206 and MRC-2, were significantly upregulated in M2 BCRT1-overexpressing or control cells. Then, the macrophages (Fig. 6a), indicating the successful conditioned medium of educated macrophages was col- polarization of monocytes. Moreover, lncRNA BCRT1 ex- lected and used to treat breast cancer cells or HUVECs. pression was elevated in M2 macrophages compared to The results showed that macrophages treated with exo- M1 macrophages (Fig. 6b), indicating a potential role of somes or supernatants isolated from lncRNA BCRT1- lncRNA BCRT1 in macrophage polarization. After PMA overexpressing groups significantly promoted cell migra- treatment for 24 h, THP-1 cells were transfected with si- tion and angiogenesis (Fig. 6j-k). Moreover, a chick NC or si-BCRT1, and then IL-4 and IL-13 were added for chorioallantoic membrane (CAM) assay revealed that 24 h to induce the M2 phenotype. The results showed that chick embryos injected with conditioned medium of M1 markers were significantly increased, while M2 educated macrophages treated with exosomes or superna- markers were remarkable decreased in the si-BCRT1 group tants isolated from lncRNA BCRT1-overexpressing (Fig. 6c). Accordingly, lncRNA BCRT1 overexpression led groups had an increase in new vessel density (Fig. 6l). to the opposite results (Fig. 6d). Moreover, the supernatant Taken together, these results suggested that lncRNA from lncRNA BCRT1-overexpressing MDA-MB-231 cells BCRT1 could be transferred through exosomes, thus caused an elevated expression of M2 markers compared to promoting M2 phenotype polarization and enhancing its that from control MDA-MB-231 cells (Fig. 6e). Then, we tumor promoting function. RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 13 of 20 Fig. 6 LncRNA BCRT1 could be secreted by breast cancer cells and promoted M2 polarization. a RT-PCR was used to detect the expression of M1 markers and M2 markers after LPS/INF-γ or IL-4/IL-13 treatment. b The expression of lncRNA BCRT1 was elevated in M2 macrophages. c M1 markers (CD80, MCP-1, iNOS, and IL-6) were significantly increased, while M2 markers (CD206 and MRC-2) were remarkably decreased in the lncRNA BCRT1 knockdown group. d LncRNA BCRT1 overexpression led to decreased expression of M1 markers and increased expression of M2 markers. e Conditioned medium derived from lncRNA BCRT1-overexpressing cells further increased the expression of M2 markers and lncRNA BCRT1 in macrophages. f Western blotting analysis of the exosomal markers CD63, Hsp70 and Hsp90 in exosomes derived from breast cancer cells with or without lncRNA BCRT1 overexpression. g Agarose gel electrophoresis and RT-PCR assays were used to detect the expression of lncRNA BCRT1 in exosomes. h Representative microscopy showing the uptake of PKH26-labeled exosomes (red fluorescent dye) derived from MDA-MB-231 cells by recipient macrophages. i The expression of M2 markers and lncRNA BCRT1 in macrophages was detected after culture with the indicated exosomes. j Cell migration was increased after cultured with lncRNA BCRT1-overexpressing exosomes or conditioned media. k-l Tube formation or CAM assays were used to evaluate the angiogenesis ability after culture with lncRNA BCRT1-overexpressing exosomes or conditioned media. (*P < 0.05, **P < 0.01, and ***P < 0.001) LncRNA BCRT1 is transcriptionally regulated by HIF-1α responsible for the aberrant expression of some under hypoxic conditions lncRNAs [32, 33]. To investigate whether lncRNA Hypoxia is one of the major intratumor characteristics BCRT1 is a hypoxia-sensitive lncRNA, breast cancer in various cancers, and several studies have revealed that cells were treated with hypoxia or normoxia for 48 h. the hypoxic microenvironment of cancers might be The results showed that the expression of lncRNA RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 14 of 20 BCRT1 was clearly elevated along with the increase in HIF-1α knockdown (Fig. 8a-c, Additional file 12: Figure HIF-1α expression (Fig. 7a-b). HIF-1α knockdown dra- S7a-c). Previous studies revealed a close association be- matically decreased HIF-1α and lncRNA BCRT1 expres- tween hypoxia and EMT; therefore, the role of lncRNA sion under both normoxic and hypoxic conditions (Fig. BCRT1 in hypoxia-induced EMT was further investi- 7c-e). Moreover, knockdown of HIF-1α substantially at- gated. After treatment with hypoxia, MDA-MB-231 cells tenuated hypoxia-induced lncRNA BCRT1 upregulation demonstrated a more fibroblast-like morphology and el- (Fig. 7c-e). To elucidate the potential mechanism of evated migration ability, which was dramatically reversed hypoxia-induced upregulation of lncRNA BCRT1, we ana- by knockdown of HIF-1α or lncRNA BCRT1 (Fig. 8d-f, lyzed the JASPAR database [34], and two putative HIF-1α Additional file 12: Figure S7d-e). Moreover, the siHIF-1α- response elements (HREs) in the lncRNA BCRT1 pro- repressed EMT profile under hypoxic conditions was moter were identified (Fig. 7f-g). To determine whether obviously rescued by overexpression of lncRNA BCRT1 HIF-1α regulates the expression of lncRNA BCRT1 (Fig. 8d-f, Additional file 12: Figure S7d-e). These results through these HREs, we constructed two luciferase re- indicated that lncRNA BCRT1 might participate in porter vectors containing the full-length lncRNA BCRT1 hypoxia-induced biological functions in breast cancer cells. promoter (HRE1 and HRE2) and a truncated fragment (HRE2). As expected, hypoxia treatment significantly in- Discussion creased the luciferase activity in cells transfected with the The therapeutic methods available to breast cancer pa- full-length lncRNA BCRT1 promoter vector compared tients with metastatic lesions are complicated, but their with the control cells, whereas the lack of HRE1 impaired clinical outcome is less than satisfactory. It is of great the luciferase activity, which suggested that HRE1 was importance to comprehensively understand the molecu- crucial for lncRNA BCRT1 transcription (Fig. 7h). In lar mechanisms involved in breast cancer metastasis and addition, HIF-1α knockdown reversed the luciferase activ- identify novel prognostic predictors. Recently, aberrant ity induced by hypoxia treatment (Fig. 7 h), suggesting that expression of lncRNAs has been reported in various can- hypoxia promoted lncRNA BCRT1 transcription through cers [9, 36], and lncRNAs have been shown to play im- HIF-1α by binding to HRE1 in its promoter region. We portant roles in tumor progression. Increasing studies performed chromatin immunoprecipitation (ChIP) assays have focused on the functions and regulation of lncRNAs with a HIF-1α antibody to further confirm the binding of to discover novel targets for the diagnosis and treatment HIF-1α with the two predicted HREs in the lncRNA of cancers. In this study, we determined that the unchar- BCRT1 promoter (Fig. 7i), and the results confirmed that acterized lncRNA BCRT1 was significantly increased in HRE1 in the lncRNA BCRT1 promoter was the major re- breast cancer tissues compared to normal tissues, and gion mediating HIF-1α-induced transcriptional regulation. high lncRNA BCRT1 expression was associated with poor Using the ChIPBase database, we found that the expres- prognosis of breast cancer patients. Functional studies re- sion of PTBP3 was positively associated with HIF-1α ex- vealed that lncRNA BCRT1 could promote the prolifera- pression (Fig. 7j). Moreover, hypoxia treatment led to tion and mobility of breast cancer cells in vitro and elevated expression of PTBP3 at the mRNA and protein in vivo, indicating a tumor-promoter role in breast cancer. levels (Fig. 7k), while HIF-1α knockdown attenuated this Although several dysregulated lncRNAs have also been effect (Fig. 7l). These results suggested that hypoxia tran- identified, more studies are needed to elucidate their scriptionally regulated lncRNA BCRT1 expression by function. HIF-1α through direct binding with HRE1 on its The biological function of lncRNAs is largely dependent promoter. on their subcellular localization. Accumulated evidence has shown that lncRNAs located in the cytoplasm could LncRNA BCRT1 mediates hypoxia-induced malignant participate in gene regulation at the posttranscriptional properties of breast cancer cells level, including by acting as ceRNAs and protecting the Hypoxia is a hallmark of the tumor microenvironment target mRNAs from repression [37, 38]. By using cell cyto- and is associated with proliferation, metastasis, and drug plasmic/nuclear fractionation and RNA FISH assays, we resistance in various solid tumors [35]. Therefore, we found that lncRNA BCRT1 was preferentially localized in first investigated whether lncRNA BCRT1 was involved the cytoplasm, indicating its potential for functioning as a in hypoxia-induced cell proliferation. Hypoxia treatment miRNA sponge. Subsequently, bioinformatics analysis in- led to increased expression of lncRNA BCRT1 and dicated that there existed binding sites of miR-1303 in the PTBP3, in accordance with enhanced cell proliferation lncRNA BCRT1 sequence, which was further validated by (Fig. 8a-c, Additional file 12: Figure S7a-c). Moreover, luciferase reporter assay and RIP assay. Moreover, the ex- HIF-1α or lncRNA BCRT1 knockdown attenuated the pression of lncRNA BCRT1 was negatively associated with effects induced by hypoxia, whereas lncRNA BCRT1 miR-1303, and a significant reciprocal repression feedback overexpression partly reversed the inhibitory effect of loop present in breast cancer cells. Importantly, miR-1303 RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 15 of 20 Fig. 7 LncRNA BCRT1 was transcriptionally regulated by HIF-1α during hypoxia. a-b The expression levels of HIF-1α protein (a) or lncRNA BCRT1 mRNA (b) in MDA-MB-231 and MDA-MB-468 cells were measured after culture under normoxia or hypoxia for 48 h by western blot or RT-PCR. c- d The efficiency of HIF-1α knockdown was detected using RT-PCR and western blot. e HIF-1α knockdown inhibited the expression of lncRNA BCRT1 in MDA-MB-231 and MDA-MB-468 cells under normoxia or hypoxia. f The recognition motif of HIF-1α from the JASPAR database. g Schematic illustration of the proximal region of the lncRNA BCRT1 promoter and the putative hypoxia responsive elements (HREs). h MDA-MB- 231 cells were transfected with a lncRNA BCRT1 promoter-containing pGL3 reporter vector and further treated with hypoxia or hypoxia combined with siHIF-1α. After 48 h, Luciferase activity was measured with the dual-luciferase reporter assay system. i ChIP assays with anti-HIF-1α antibody were performed to verify the binding between HIF-1α and the HREs of the lncRNA BCRT1 promoter under normoxia and hypoxia. j PTBP3 and HIF-1α expression from the TCGA breast cancer dataset was analyzed by the starBase database. k The mRNA and protein expression of PTBP3 was elevated under hypoxic conditions. l After HIF-1α knockdown, the expression of PTBP3 was evaluated by RT-PCR and western blot in MDA-MB-231 and MDA-MB-468 cells under normoxia or hypoxia. (*P < 0.05, **P < 0.01, and ***P < 0.001) RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 16 of 20 Fig. 8 LncRNA BCRT1 is essential for HIF-1α-mediated hypoxia-induced malignant properties. MDA-MB-231 cells were treated with normoxia, hypoxia, a combination of siHIF-1α and hypoxia, a combination of si-BCRT1 and hypoxia, or hypoxia and cotransfection with siHIF-1α and the pcDNA3.1-BCRT1 plasmid. a The expression of HIF-1α and PTBP3 was assessed by western blot. b The expression of lncRNA BCRT1 and PTBP3 was evaluated by RT-PCR. c Colony formation assay was used to evaluated the proliferation of MDA-MB-231 cells. d Monolayer morphology of MDA-MB-231 cells were photographed. e-f Transwell assay and wound healing assay were used to analyze the migration ability of MDA-MB-231 cells. (*P < 0.05, **P < 0.01, and ***P < 0.001) acted as a tumor suppressor in breast cancer, and miR- behavior of various cancers. PTBP3 was found to be up- 1303 overexpression partially reversed lncRNA BCRT1 regulated in gastric cancer compared with normal gastric overexpression-mediated promotion of proliferation, mi- mucosa [40], and high PTBP3 expression was correlated gration, invasion, and angiogenesis of breast cancer cells. with poor prognosis and higher lymph node metastasis Together, our results revealed that lncRNA BCRT1 could in gastric cancer patients. Further study revealed that serve as a ceRNA by sponging miR-1303 in breast cancer. PTBP3 was positively associated with metastasis of gas- Polypyrimidine tract-binding protein 3 (PTBP3), an tric cancer by regulating CAV1 through alternative spli- essential RNA-binding protein with roles in RNA alter- cing [25]. Moreover, a prooncogenic role for PTBP3 has native splicing (AS) [39], plays an important role in also been discovered in hepatocellular carcinoma medi- regulating gene expression and affects the biological ated by regulation of the splicing balance of NEAT1 and RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 17 of 20 pre-miR-612 [26]. In addition, previous studies reported 30–100 nm vesicles, can be secreted by cancer cells and in- that PTBP3 knockdown led to increased apoptosis and fluence tumor progression or drug resistance by modulat- cell cycle arrest, either through regulation of p53 signal- ing other cells in the microenvironment via intercellular ing [41] or through HDAC6-mediated inhibition of the communication [46]. Various exosomal lncRNAs have been phosphorylation of Akt and thymidylate synthase reported to participate in intercellular communication and (TYMS) expression [42]. However, the physiological are associated with the diagnosis and prognosis of cancer roles or molecular functions of PTBP3 in breast cancer [47]. Our results revealed that breast cancer cell-derived remain largely unclear, except one study that reported exosomes could promote M2 polarization and enhance its that PTBP3 promoted cell proliferation, migration, and tumor-promoting function by transmitting lncRNA invasion of breast cancer cells by preventing ZEB1 BCRT1. Nevertheless, the correlation between exosomal mRNA degradation [43]. However, the regulatory mech- lncRNA BCRT1 expression and the diagnostic or prognos- anism involved in the expression and function of PTBP3 tic values in breast cancer still needs further investigation. in breast cancer has not been fully elucidated. In the Hypoxia is a common phenomenon in various cancers current study, we identified PTBP3 as a target protein of and is associated with cancer progression. Several the lncRNA BCRT1/miR-1303 axis on the basis of the lncRNAs have been reported to be regulated by hypoxia following observations. Through bioinformatic predic- via HIF-1α-mediated transcriptional regulation. These tion and dual-luciferase reporter assays, PTBP3 was hypoxia-sensitive lncRNAs, such as lncRNA PVT1 [48], demonstrated to be a direct target gene of miR-1303 in lncRNA HITT [49], and LncRNA-MTA2TR [50], partici- breast cancer cells. Moreover, lncRNA BCRT1 overex- pate in tumorigenesis and tumor metastasis. In our study, pression led to increased expression of PTBP3, which we identified lncRNA BCRT1 as a hypoxia-sensitive could be partially reversed by miR-1303 overexpression, lncRNA. Using the JASPAR database, we predicted two indicating a lncRNA BRCT1/miR-1303/PTBP3 axis in potential HREs in the promoter of lncRNA BCRT1. More- breast cancer. We also revealed a significant positive re- over, the expression of lncRNA BCRT1 was increased lationship between the expression of lncRNA BCRT1 during hypoxia, which could be repressed by HIF-1α and PTBP3 in breast cancer cells. Furthermore, we re- knockdown. Furthermore, ChIP and dual-luciferase re- vealed that PTBP3 was increased in breast cancer tissues porter assays verified the regulatory effect of HIF-1α on and that PTBP3 knockdown clearly inhibited the prolif- lncRNA BCRT1 transcription in response to hypoxia. eration, migration and invasion of breast cancer cells. Notably, our results showed that hypoxia led to increased Hence, we further demonstrated the oncogenic role of expression of PTBP3 and lncRNA BCRT1 knockdown re- PTBP3 and provided evidence for the posttranscriptional pressed hypoxia-induced PTBP3, while lncRNA BCRT1 regulation of PTBP3 by a lncRNA in breast cancer. overexpression partially reversed the inhibition of PTBP3 Recently, considerable attention has been focused on the expression by HIF-1α knockdown. These results indicated significance of the tumor environment on tumor progres- a novel indirect pathway for hypoxia-induced PTBP3 ex- sion, a complex community that includes cancer cells, pression that was stimulated by increased lncRNA BCRT1 cancer-associated fibroblasts (CAFs), and immune inflam- levels. Moreover, our results revealed that lncRNA BCRT1 matory cells [44]. The interaction between cancer cells and knockdown could suppress hypoxia-induced proliferation TAMs, one of the most abundant immune cells in various and migration, whereas lncRNA BCRT1 overexpression solid cancers, was correlated with tumor progression, drug could rescue these effects, which was repressed by HIF-1α resistance, and poor prognosis in cancer patients [45]. knockdown under hypoxic conditions. Therefore, our Based on their biological properties, macrophages are study provided novel evidence supporting lncRNA as a generally categorized into two major phenotypes, pro- link between hypoxia and cancer progression. inflammatory (M1) and anti-inflammatory (M2) macro- phages. Many studies have demonstrated that TAMs are Conclusions considered M2-like macrophages that are closely associated In summary, we identified hypoxia-responsive lncRNA with cancer progression. Our results showed that lncRNA BCRT1 as a tumor-promoter in breast cancer, and the BCRT1 was increased in M2-like macrophages compared higher expression of lncRNA BCRT1 was associated with to M1-like macrophages and unpolarized macrophages. tumor metastasis and poor prognosis. LncRNA BCRT1 Moreover, lncRNA BCRT1 overexpression remarkably pro- acted as a sponge for miR-1303 to attenuate its repressive moted the expression of markers of M2-like macrophages, effect on PTBP3 and promoted M2 polarization through whereas lncRNA BCRT1 knockdown produced the oppos- exosome-mediated transfer. Our results provide a better ite results, indicating a promoting role of lncRNA BCRT1 understanding of the role of lncRNAs in breast cancer in M2 polarization. The conditioned medium of breast can- progression and a potential therapeutic target and prog- cer cells could influence the polarization of macrophages, nostic predictor against this malignancy. indicating the existence of a transfer mediator. Exosomes, RETRACTED ARTICLE Liang et al. Molecular Cancer (2020) 19:85 Page 18 of 20 Supplementary information detect the expression of TGFβ in macrophages after indicated treatment. Supplementary information accompanies this paper at https://doi.org/10. (**P < 0.01, ***P < 0.001, Student’s t test). 1186/s12943-020-01206-5. Additional file 12: Figure S7. LncRNA BCRT1 is essential for HIF-1α- mediated hypoxia-induced malignant properties. MDA-MB-468 cells were Additional file 1: Table S1. Primers used for RT-PCR and vector treated with normoxia, hypoxia, a combination of siHIF-1α and hypoxia, a construction. combination of si-BCRT1 and hypoxia, or hypoxia further and cotransfec- Additional file 2: Table S2. Antibodies used in the experiments. tion with siHIF-1α and the pcDNA3.1-BCRT1 plasmid. a The expression of HIF-1α and PTBP3 was evaluated by western blot. b RT-PCR was used to Additional file 3: Figure S1. The sequence, secondary structure and detect the expression of lncRNA BCRT1 and PTBP3. c Proliferation of coding capacity of lncRNA BCRT1. a Schematic diagram showing the MDA-MB-468 cells was assessed by colony formation assay. d-e Transwell genomic locus of lncRNA BCRT1 in humans. Pink rectangles represent assay and wound healing assay were applied to analyze the migration exons. b The sequence of lncRNA BCRT1. c The secondary structure of ability of MDA-MB-468 cells. (*P < 0.05, **P < 0.01, and ***P < 0.001). lncRNA BCRT1 from AnnoLnc (http://annolnc.cbi.pku.edu.cn/). d Putative ORFs of lncRNA BCRT1 were predicted by the ORF Finder. e The amino acid sequences of the putative proteins. f The coding potential of lncRNA Abbreviations BCRT1 was measured by 5 different metrics and the results showed that LncRNAs: Long non-coding RNAs; GEO: Gene Expression Omnibus; lncRNA BCRT1 had no coding potential. TCGA: The Cancer Genome Atlas; ceRNA: competing endogenous RNA; NC: Negative control; EMT: Epithelial–mesenchymal transition; RIP assay: RNA Additional file 4: Table S3. Correlation between LncRNA BCRT1 immunoprecipitation assay; ChIP: Chromatin immunoprecipitation expression and clinicopathological features in breast cancer patients. Additional file 5: Table S4. Univariate analysis of overall survival in Acknowledgements breast cancer patients (n = 68). Not applicable. Additional file 6: Table S5. Multivariate analysis of overall survival in breast cancer patients (n = 68). Authors’ contributions Additional file 7: Figure S2. LncRNA BCRT1 regulates proliferation and YRL and QFY conceived the study; YRL, XJS, YML, HWZ, YL, and DWH migration of breast cancer cells in vitro. a The efficiency of lncRNA BCRT1 performed the experiments; BC, WJZ, NZ, TTM, LJW, and XYL collected knockdown in MCF-7 cells was validated with RT-PCR. b MTT assays clinical samples; YJW, FZY, and DL analyzed the data; YRL and XJS wrote the showed the reduced proliferation of MCF-7 cells transfected with si- paper; YRL and QFY revised the paper. All authors read and approved the BCRT1. c Colony formation assay showed the decreased proliferation of final manuscript. MDA-MB-231 and MDA-MB-468 cells after lncRNA BCRT1 knockdown. d The proliferation rate of MCF-7 cells was evaluated after lncRNA BCRT1 Funding overexpression. e-f MTT assay and colony formation assay were used to This work was supported by National Natural Science Foundation of China evaluate proliferation rate after lncRNA BCRT1 overexpression in MCF-7 (No.81272903; No.81672613; No.81874119; No. 81502285; No.81602329), cells. g-h Transwell assays demonstrated that lncRNA BCRT1 knockdown China Postdoctoral Science Foundation (No. 2018 M630787), Shandong inhibited whereas lncRNA BCRT1 overexpression promoted cell migration Science and Technology Development Plan (No. 2016CYJS01A02) and and invasion abilities in MCF-7 cells. (**P < 0.01, ***P < 0.001, Student’st Special Support Plan for National High-Level Talents (Ten Thousand Talents test). Program W01020103). Additional file 8: Figure S3. LncRNA BCRT1 and miR-1303 could mutu- Availability of data and materials ally regulate each other and miR-1303 overexpression inhibited cell prolif- The datasets used and/or analyzed during the current study are available eration and metastasis in vitro. a RT-PCR was used to validate the change from the corresponding author on reasonable request. of miR-1303 levels after lncRNA BCRT1 overexpression in MDA-MB-231 and MDA-MB-468 cells. b LncRNA BCRT1 expression was increased in Ethics approval and consent to participate tumor tissues from lncRNA BCRT1-overexpressing group compared to This project was approved by the Ethical Committee on Scientific Research control group. MiR-1303 expression in tumor tissues from lncRNA BCRT1- of Shandong University Qilu Hospital. overexpressing group was lower than those from control group. c Over- expression of miR-1303 was associated with better overall survival of Consent for publication breast cancer patients according to the LinkedOmics databases. d The ef- All human tissue samples were obtained with written informed consent ficiency of of miR-1303 overexpression in MCF7 cells was validated by RT- from all subjects. PCR. e MTT assays showed the reduced proliferation in miR-1303- overexpresing MCF-7 cells. f Transwell migration assays demonstrated Competing interests that miR-1303 overexpression inhibited cell migration. Columns are the The authors declare that they have no competing interests. average of three independent experiments. g LncRNA BCRT1 expression was decreased in MDA-MB-231 and MDA-MB-468 cells transfected with Received: 24 January 2020 Accepted: 23 April 2020 miR-1303 mimics. 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Journal

Molecular CancerSpringer Journals

Published: May 8, 2020

Keywords: LncRNA BCRT1; miR-1303; PTBP3; Progression; Breast cancer

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