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Long non coding RNA OIP5-AS1 promotes metastasis of breast cancer via miR-340-5p/ZEB2 axis

Long non coding RNA OIP5-AS1 promotes metastasis of breast cancer via miR-340-5p/ZEB2 axis ONCOLOGY REPORTS 44: 1662-1670, 2020 Long non coding RNA OIP5‑AS1 promotes metastasis of breast cancer via miR‑340‑5p/ZEB2 axis 1 2 1 3 LINGJUN MENG , XIAOJING YUE , DI ZHOU and HONGJUN LI Department of Hematology and Oncology, China‑Japan Union Hospital Affiliated to Jilin University, Changchun, Jilin 130033; Department of Developmental and Behavioral Pediatrics, The First Hospital of Jilin University, Changchun 130021; Health Management Medical Center, China‑Japan Union Hospital Affiliated to Jilin University, Changchun, Jilin 130033, P.R. China Received January 22, 2020; Accepted July 20, 2020 DOI: 10.3892/or.2020.7724 Abstract. Breast cancer is the most common invasive cancer Introduction in women with the highest number of related deaths which is caused by distal metastasis. Recently, integrated analysis of Currently, breast cancer remains a leading health problem and gene expression profile suggested widespread gene dysregula - constitutes one of the most severe burdensome diseases in tion in various types of cancer. Research in the past decade females around the world despite understanding of underlying has focused on long non-coding RNAs (lncRNAs), particu- molecular mechanisms (1). Tumor metastasis is diagnosed larly in cell proliferation, tumor progression and metastasis. in approximately 30% of breast cancer patients and is the OPA-interacting protein 5 antisense transcript 1 (OIP5-AS1) major cause of cancer-related deaths (2). The prognosis for is an evolutionarily conserved long non-coding RNA that most patients with metastatic breast cancer is unfavorable has been linked to oncogenesis in multiple cancers. In breast with a median overall survival range from 2 to 3 years (3). cancer, dysregulation of OIP5-AS1 was reported but the Generally, breast cancer can be categorized into four subtypes precise role in cancer development and progression remains [luminal A, luminal B, human epidermal growth factor unclear. In the present study, using small interfering RNA receptor 2 (HER2) positive and triple negative], which are (siRNA) targeting OIP5-AS1, it was shown that knockdown defined using immunohistochemical breast tumor markers (4). of OIP5-AS1 was associated with alteration of EMT markers These four subtypes have the potential risk of distant metas- and suppressed migration and invasion of breast cancer tasis but with differential site‑specic fi metastatic patterns (5). cells. Among the EMT-related transcription factors, ZEB1 Currently, breast cancer metastasis from primary tumor to and ZEB2 were signic fi antly downregulated with OIP5‑AS1 distant organs occurs through a sequential molecular cascade knockdown. Computational analysis and a dual-luciferase including local angiogenesis for tumor growth, invasion of the reporter system identified miR‑340‑5p was the target gene surrounding tissue, intravasation of the carcinoma cells into for OIP5‑AS1. Further experiments verified the function of the blood or lymphatic vessels, dissemination and proliferation OIP5‑AS1 in cell invasion was dependent on miR‑340a‑5p at secondary neoplastic foci (6). These carcinoma cells obtain through regulating target gene ZEB2. In vivo study demon- mesenchymal features and suppress their epithelial features strated that overexpressing OIP5-AS1 in breast cancer cells through the epithelial-to-mesenchymal transition (EMT) promoted lung metastasis in nude mice. The findings of the process to promote an invasive and metastatic phenotype (7). present study revealed the mechanism of OIP5-AS1 in breast Multiple transcription factors coordinate EMT programs. cancer metastasis. Overall, our study may provide a potential Among them, zinc‑finger E‑box‑binding (ZEB) transcription therapeutic target for breast cancer metastasis. factors, ZEB1 and ZEB2, are two EMT regulators that either repress or activate transcription in various types of cancer (8). Furthermore, ZEB2 was reported to negatively correlate with the epithelial marker E-cadherin in breast cancer cells involved in breast cancer progression (9). Long non-coding RNAs (lncRNAs) are a class of tran- Correspondence to: Professor Hongjun Li, Health Management scripts containing more than 200 nucleotides in length with Medical Center, China-Japan Union Hospital Affiliated to limited protein‑coding capacity (10). Recent findings have Jilin University, 126 Xiantai Street, Changchun, Jilin 130033, shown that dysregulation of lncRNAs is involved in cell prolif- P.R. China eration, tumor progression and metastasis in cancers (11). E-mail: hj_li@jlu.edu.cn Functionally, lncRNAs interact with proteins and other RNAs Key words: OIP5‑AS1, miR‑340‑5p, ZEB2, breast cancer, to regulate their activities and cellular location. Furthermore, metastasis lncRNAs act as molecular sponges for miRNAs that block the binding activity for target transcripts (12). In breast cancer, several lncRNAs have been identified as either oncogenic MENG et al: OIP5-AS1 PROMOTES BREAST CANCER METASTASIS VIA miR‑340‑5p/ZEB2 AXIS or tumor suppressive factors, such as X-inactive-specific- non-coated chambers with a pore size of 0.8 µM. The trans- transcript (XIST), HOX antisense intergenic RNA (HOTAIR), fected cells were seeded into the upper chamber in DMEM growth arrest specic fi 5 (GAS5) and metastasis‑associated lung with 1% FBS and the lower chamber was filled with 10% FBS adenocarcinoma transcript 1 (MALAT1) (13‑16). A systematic as a chemoattractant. After 24‑h incubation in the humidie fi d analysis of the correlation has been carried out between these incubator at 37˚C with 5% CO , cells in the upper chamber dysregulated lncRNAs and breast cancer clinicopathology and were removed and cells in the lower side were fixed with survival suggesting a pivotal role in cancer development (17). 4% PFA and stained by 1% crystal violet. Stained cells were Increasing lncRNAs have been shown to participate in specic fi then visualized and imaged at a x20 magnic fi ation by a light cancer types but more often exert general function in a broad microscope. spectrum of cancer. OPA-interacting protein 5 antisense transcript 1 (OIP5-AS1) Western blot analysis. The cellular proteins were extracted is an evolutionarily conserved long non-coding RNA that is in RIPA buffer supplemented with protease inhibitor. The transcribed from opposite direction to the OIP5 gene. It was protein concentration was quantie fi d using BCA protein assay first shown to be expressed in the nervous system and was and 20 µg of each protein sample was loaded and analyzed by essential for neurogenesis during embryonic development (18). 10% sodium dodecyl sulfate (SDS)‑polyacrylamide gel elec- The functions of OIP5-AS1 in multiple human cancers have trophoresis (PAGE) system. Then, proteins were transferred to been reported to be associated with oncogenesis (19,20). a PVDF membrane. The membrane was blocked in 5% BSA In breast cancer, OIP5-AS1 levels are upregulated in breast and probed with primary antibodies: Anti‑ZEB2 (1:1,000; tumor tissue and correlated with tumor size, metastatic status no. ab138222, Abcam), anti‑E‑cadherin (1:1,000; no. 14472, of lymph nodes, pathological grading and TNM stage (21). Cell signaling), anti‑N‑cadherin (1:1,000; no. ab18203, Abcam), In the present study, we investigated the role of OIP5-AS1 anti‑vimentin (1:1,000; no. ab92547, Abcam), anti‑ZEB1 in breast cancer metastasis using the in vitro and in vivo (1:1,000; no. 70512, Cell signaling), anti‑Snail (1:1,000; models showing that OIP5-AS1 regulates ZEB2 expression by no. IMG‑6639A, Novus Biologicals), anti‑Slug (1:1,000; acting as ceRNA for miR‑340‑5p. no. 9585, Cell signaling), anti‑Twist (1:1,000; no. 69366, Cell signaling) and anti‑ GA PDH (1:2,0 0 0; no. ab8245, Materials and methods Abcam). Then, the membrane was incubated with peroxi- dase-conjugated anti-mouse or anti-rabbit secondary antibody Cell culture and transfection. Breast cancer cell lines MCF-7, (1:2,000, nos. NEF822001EA; NEF812001EA, PerkinElmer). MDA‑MB‑231, ZR‑75, MDA‑MB‑468, SKBR3 and normal Immunoreactivity bands were detected by chemiluminescence human epithelial cell line MCF-10A were purchased from the and the intensity of the bands was quantie fi d using Image Lab American Type Culture Collection (ATCC). MCF-10A cells Software (Bio Rad, China). were cultured in MEBM (Lonza) and supplemented with 100 ng/ml cholera toxin. ZR‑75 and SKBR3 cell lines were RNA fluorescence in situ hybridization (FISH). A Cy3‑labeled maintained in RPMI‑1640 medium (Sigma) containing 10% set of probes recognizing OIP5-AS1 was designed and synthe- FBS, 2 mM L‑glutamine and 2% penicillin and streptomycin. sized by Biosearch Technologies. The MCF-7 cells were MCF‑7, MDA‑MB‑231, and MDA‑MB‑468 cell lines were cultured on coverslips for 24 h and then fixed in 4% PFA. After maintained in DMEM supplemented with 10% FBS plus permeabilization with 70% ethanol at 4˚C for 1 h, cells were 2% penicillin and streptomycin. Cells were cultured in a hybridized with the OIP5-AS1 probes dissolved in hybridiza- humidie fi d incubator at 37˚C with 5% CO . tion buffer (no. SMF-HB1-10, Biosearch Technologies) at The pre-designed siOIP5-AS1 and siZEB2 were 37˚C in the dark for 16 h. The nucleus was stained with DAPI. purchased from ThermoFisher (no. 4390771, no. AM16708) Images were captured using a confocal microscope (Olympus). and transfected into cells using Lipofectamine RNAiMAX reagent (ThermoFisher) following the manufacturer's Quantitative real‑time PCR (RT‑PCR). Total RNA was instructions. miR‑340‑5p mimics, negative control mimics, ext r a ct e d f rom c el ls ( MC F‑7, M DA‑M B ‑231, Z R‑75, miR‑340‑5p inhibitors and negative control inhibitors were MDA‑MB‑468, SKBR3, and MCF‑10A) using TRIzol reagent purchased from GenePharma and transfected into cells using (Thermo Fisher). Total RNA (1 µg) was reverse transcribed Lipofectamine 2000 reagent (ThermoFisher), according to the into cDNA and a SYBR-Green quantitative real-time PCR manufacturers' instructions. miR‑340‑5p mimic: 5'‑UUA UAA Master Mix kit was used to detect qPCR signals. The targeted AGC AAU GAG ACU GAU U‑3' and miR‑340‑5p inhibitor: gene expression was normalized with GAPDH and calcu- - Cq ΔΔ 5'‑AAU CAG UCU CAU UGC UUU AUA A‑3'. Cells were used lated using 2 method (22). The primer sequences used for further experiments at 48 h after transfection. were: OIP5‑AS1: 5'‑TGC AAC CCA AGG TGG ATA CT‑3' and 5'‑GAG AGA CTG CAG TGA GCA GA‑3'; ZEB2: 5'‑CAG CTC Wound healing assay. MCF‑7 and MDA‑MB‑231 cells were TTC CAC CTC AAA GC‑3' and 5'‑TCC TTG TTT CCG CTG seeded in 12-well plates and transfected with either siNC or GTA CT‑3'; GAPDH: 5'‑GT C GGA GTC AAC GGA TTT GG‑3' siOIP5-AS1. A linear wound was scratched across the center of and 5'‑TGA CGG TGC CAT GGA ATT TG‑3'. For the detection the well using a sterile pipette tip. The images of wound closure of miR‑340‑5p, stem‑loop qRT‑PCR was performed using were captured after 24 h using Olympus microscope (x10). miScript SYBR‑Green PCR Kit with U6 small nuclear RNA as an internal control (Qiagen). The following thermocycling Transwell invasion assay. The invasion of MCF-7 and conditions were used in the experiments: PCR initial activa- MDA‑MB‑231 cells was detected using matrigel‑coated or tion at 95˚C for 15 min, followed by 40 cycles of denaturation ONCOLOGY REPORTS 44: 1662-1670, 2020 at 94˚C for 15 sec, annealing at 55˚C for 30 sec and an exten- with LV-OIP5-AS1 or LV-NC were intravenously injected sion at 70˚C for 30 sec. through the tail vein of BALB/c nude mice under isou fl rane anaesthesia. After 8 weeks of inoculation, the mice were RNA immunoprecipitation assay. Magna RIP kit (Millipore) euthanized and the number of lung metastatic tumors per lung was used for RNA immunoprecipitation experiments. The were counted under a dissecting microscope and confirmed by procedure was performed following the manufacturer's H&E staining. The experimental protocols were approved by protocol. Brief ly, after miR‑340‑5p mimics or NC mimics the Animal Care Committee of China-Japan Union Hospital transfection, the cells were lysed in RIP lysis buffer. The cell Affiliated to Jilin University. lysate was incubated with either Ago2 antibody or control IgG together with protein A/G magnetic beads. Then the beads Statistical analysis. Data are expressed as mean ± standard were washed and incubated with Proteinase K at 55˚C for error mean (SEM). The Student's t test was employed to 30 min to digest proteins. The purie fi d RNA was obtained and compare two groups and one-way ANOVA with post hoc analyzed by RT-qPCR. test was used to analyze differences among multiple groups. A value of P<0.05 was considered as statistically signic fi ant. Dual‑luciferase reporter assay. In this study, the OI P5 ‑AS1/m i R NA i nt er a ct ions were pre d ict e d usi ng Results Starbase (http://starbase.sysu.edu.cn/) and DIANA‑LncBase database (http://www.microrna.gr/LncBase). For OIP5‑AS1 Interference of OIP5‑AS1 represses epithelial‑to‑mesenchymal a nd m i R‑34 0 ‑5p binding activit y, OI P5‑AS1 f ragment transition (EMT) in breast cancer cells by regulating ZEB containing the binding sites of miR‑340‑5p, as well as those family proteins. The dysregulation of long non-coding RNA of the wild-type and mutant sequences were cloned into a OIP5-AS1 was involved in multiple cancer types associating pmirGLO Dual-luciferase Vector designated as OIP5-AS1 with overall survival, TNM stage and prognosis (21,23‑25). In WT or OIP5‑AS1 MUT. For ZEB2 and miR‑340‑5p binding breast cancer, studies reported that OIP5-AS1 is upregulated activity, fragment of 3'UTR ZEB2 containing the binding in both tumor samples and cell lines (21). We first evaluated sites of miR‑340‑5p, as well as the wild‑type and mutant the expression levels of OIP5‑AS1 in v fi e breast cancer cell sequences were cloned into a pmirGLO Dual-luciferase lines. The results showed that the relative expression levels of Vector designated as ZEB2 WT or ZEB2 MUT. These vectors OIP5‑AS1 were much higher in the v fi e breast cancer cell lines were co‑transfected with either NC mimics or miR‑340‑5p than in the normal epithelial cell line MCF-10A (Fig. 1A). mimics using Lipofectamine 2000 reagent. At 48 h after Then, we chose luminal-type breast cancer cell line MCF-7 transfection, the relative luciferase activities were recorded and basal B TNBC cell line MDA‑MB‑231 for further func- by dual- luciferase reporter assay system (Promega) and the tional studies. To investigate the role of OIP5-AS1 in breast values were normalized to the Renilla luciferase activity. cancer metastasis, we efficiently knocked down OIP5‑AS1 with siRNAs in MCF‑7 and MDA‑MB‑231 cell lines (Fig. 1B) Immunohistochemistry staining and hematoxylin and eosin and analyzed the cell migration and invasion properties. In (H&E) staining. The lung of nude mice was dissected and the wound healing assay, siOIP5-AS1 groups showed a slower fixed in 10% formaldehyde at room temperature overnight. migration rate than the siNC group (Fig. 1C). Furthermore, The embedded samples in paraffin were sectioned into 5 µm knockdown of OIP5‑AS1 in the two cell lines significantly slices and mounted on glass slides. For immunohistochemical inhibited cell invasion (Fig. 1D). Next, we assessed the effects staining, the slides of interest were probed with anti‑Ki‑67 of the downregulation of OIP5-AS1 on the expression of antibodies (1:500; Abcam) and then the secondary streptav- epithelial-to-mesenchymal transition (EMT) markers. The idin-horseradish peroxidase-conjugated antibody staining. protein analysis results indicated that the epithelial marker Immunoreactivity was visualized by DAB and lightly E-cadherin was increased whereas the mesenchymal markers counterstained with 5% hematoxylin. For H&E staining, N-cadherin and Vimentin were decreased (Fig. 1E). These slides were deparaffinized and rehydrated in graded ethanol results suggested that downregulation of OIP5-AS1 repressed solutions, then in distilled water. After H&E staining, slides epithelial-to-mesenchymal transition (EMT). Considering the were mounted and examined under a light microscope. importance of transcription factors in EMT co-ordination, we further tested the expression of EMT-related transcrip- Lentivirus production and in vivo metastasis assay. Full-length tion factors (ZEB1, ZEB2, Snail, Slug and Twist). In the cDNA of human OIP5‑AS1 was amplie fi d from the mRNA of siOIP5‑AS1 group, ZEB1 and ZEB2 were signic fi antly down - MCF‑7 cells and subcloned into pcDNA3.1 (AddGene). The regulated whereas the expression of Snail, Slug and Twist were lentiviral and packaging vectors (AddGene) were co-transfected not affected (Fig. 1F). Thus, we speculated that OIP5-AS1 into HEK293FT cells using Lipofectamine 2000 reagent may exert functions in EMT through regulating ZEB family (ThermoFisher) according to the manufacturer's instructions. proteins. Virus was collected and concentrated at 48 h after transfection. Twenty healthy 6‑ to 8‑week‑old female BALB/c nude mice OIP5‑AS1 directly targeted miR‑340 ‑5p. LncRNAs exert (The Animal Institute, Jilin University) were used in this study function in various aspects of cellular function and biological and randomly divided into two groups. The mice were housed process in either nucleus or cytoplasm. In nucleus, lncRNAs in a specific pathogen‑free (SPF) facility and exposed to a may take part in chromatin remodeling and modic fi ation or 12‑h light/dark cycle. Water and food were offered ad libitum. gene expression prior to transcription, whereas lncRNAs After 1 week of acclimatization, MCF-7 cells (1x10 ) infected in cytoplasm mainly participate in post-transcriptional MENG et al: OIP5-AS1 PROMOTES BREAST CANCER METASTASIS VIA miR‑340‑5p/ZEB2 AXIS Figure 1. Downregulation of OIP5-AS1 repressed migration, invasion and epithelial-to-mesenchymal transition in breast cancer cells. (A) RT-qPCR anal- ysis of OIP5-AS1 expression in breast cancer cell lines compared with normal mammary MCF-10A epithelial cells. P<0.05. (B) RT-qPCR analysis of ** OIP5‑AS1 expression with siNC or siOIP5‑AS1 transfection in MCF‑7 and MDA‑MB‑231 cells. P<0.01 compared with siNC. (C) Migration of MCF-7 and MDA‑MB‑231 cells in siNC or siOIP5‑AS1 transfection groups was determined by wound healing assay. siOIP5‑AS1 resulted at a slower closing rate. (D) Transwell invasion assay demonstrated less invaded cells with siOIP5‑AS1 transfection in MCF‑7 and MDA‑MB‑231 cells. (E) The protein expression of EMT markers (E-cadherin, N-cadherin and vimentin) indicated the suppression of epithelial-to-mesenchymal transition with siOIP5-AS1 transfection in MCF‑7 and MDA‑MB‑231 cells. GAPDH was used as an internal control. (F) The protein level of EMT‑related transcriptional factors (ZEB2, ZEB1, Snail, Slug and Twist) in siNC or siOIP5-AS1 transfection groups indicating the selective regulation of ZEB2 and ZEB1. GAPDH was used as an internal control. P<0.05. NC, negative control. regulation and post‑translational modic fi ation (26,27). Thus, was mainly localized in the cytoplasm in MCF-7 cells which we assessed subcellular location of OIP5-AS1 by fluores- indicated OIP5-AS1 may serve as a ceRNA in breast cancer cence in situ hybridization (FISH). The detected OIP5-AS1 cells (Fig. 2A). ONCOLOGY REPORTS 44: 1662-1670, 2020 Figure 2. OIP5‑AS1 directly targeted miR340‑5p. (A) RNA FISH probed endogenous OIP5‑AS1 (red) was located in cytoplasm in MCF‑7 cells. Scale bar, 10 µm. (B) RNA‑IP analysis confirmed the direct binding of miR‑340‑5p mimics and OIP5‑AS1 in MCF‑7 cells. The expression of OIP5‑AS1 was detected using RT-qPCR. P<0.05. (C) The predicted binding sites of miR‑340‑5p on OIP5‑AS1, and target sequences were mutated. (D) Luciferase activity of MCF‑7 ** cells co‑transfected with OIP5‑AS1 WT or OIP5‑AS1 MUT and miR‑340‑5p mimics or NC mimics. P<0.01 compared with OIP5-AS1 WT and NC mimics ** group. (E) Stem‑loop RT‑qPCR analysis of miR‑340‑5p with siNC or siOIP5‑AS1 transfection in MCF‑7 and MDA‑MB‑231 cells. P<0.01 compared with siNC group. In this study, the OIP5‑AS1/miRNA interactions were expression of miR‑340‑5p in breast cancer cell lines. The predicted using Starbase and DIANA tools. Among the level of miR‑340‑5p was decreased in MCF‑7, MDA‑MB‑231, predicted miRNAs, miR‑340‑5p possesses three target sites ZR‑75, MDA‑MB‑ 468 and SKBR3 cells as compared to on OIP5‑AS1. In order to confirm that miR‑340‑5p is the human breast epithelial cell line MCF10A (Fig. 3A). target gene of OIP5-AS1, we per for med anti-Ago2 RIP assay Next, we screened mRNA targets of miR‑340‑5p using and dual luciferase reporter assay. In anti-Ago2 RIP assay, TargetScan and Starbase tools and found the 3'UTR of the endogenous OIP5-AS1 was specifically enriched in ZEB2 mRNA contains two binding sites for miR‑340‑5p. miR‑340‑5p mimics‑transfected cells when compared with We transfected miR‑340‑5p mimics into MCF‑7 cells and NC mimics group (Fig. 2B). We constructed the OIP5-AS1 detected the expression of ZEB2 mRNA by RT-qPCR. With wild-type and mutant reporter plasmids according to the the miR‑340‑5p overexpression (Fig. 3B), the level of ZEB2 bi ndi ng sequences of m i R‑34 0 ‑5p ( Fig. 2C). T he dua l m R NA was decreased (Fig. 3C). We fur ther confir med luciferase reporter assay showed that the reduced lucif- the direct binding between ZEB2 mRNA and miR‑340‑5p erase activity was only found in the miR‑340‑5p mimics by dual luciferase reporter assay. The ZEB2 3'UTR was and OIP5-AS1 wild-type co-transfection groups but not constructed and the mutant form was designed according to in the OIP5-AS1 mutant co-transfection group (Fig. 2D). the miR‑340‑5p binding sequences (Fig. 3D). As shown in Moreover, we tested the expression of m iR‑340 ‑5p with Fig. 3E, the luciferase activity was only reduced in the ZEB2 OIP5-AS1 knockdown in breast cancer cells. After two 3'UTR wild‑type and miR‑340‑5p mimics co‑transfection days with siOIP5‑AS1 transfection, the level of miR‑340‑5p group which suggested the direct binding between ZEB2 was increased in MCF‑7 and MDA‑MB‑231 cells (Fig. 2E). m R NA and m iR‑340 ‑5p. In addition, we exam ined the Taken together, these results confirmed the direct binding effects of miR‑340‑5p on the protein expression of ZEB2 by act ivit y bet we en OI P5 ‑AS1 a nd m i R‑34 0 ‑5p i n breast overexpression of either miR‑340‑5p mimics or inhibitors in cancer cells. MCF-7 cells. Similarly, the level of ZEB2 was decreased with miR‑340‑5p mimics transfection whereas it was increased miR‑340 ‑5p is downregulated in breast cancer cells and with miR‑340‑5p inhibitors transfection (Fig. 3F and G). regulates ZEB2 expression. A recent study reported that Collectively, these results supported that miR‑340‑5p regulates miR‑340‑5p was negatively associated with distant metastasis ZEB2 expression by binding to complementary sequences in in invasive breast cancers (28). Thus, we measured the relative the 3'UTR of ZEB2 mRNA. MENG et al: OIP5-AS1 PROMOTES BREAST CANCER METASTASIS VIA miR‑340‑5p/ZEB2 AXIS Figure 3. miR‑340‑5p is downregulated in breast cancer cells and regulates ZEB2 expression. (A) RT‑qPCR analysis of miR‑340‑5p expression in breast cancer cell lines compared with normal mammary MCF-10A epithelial cells. P<0.05. (B) The relative expression of miR‑340‑5p in MCF‑7 cells transfected with ** miR‑340‑5p mimics. P<0.01 compared with NC mimics group. (C) RT-qPCR analysis of relative ZEB2 mRNA expression in MCF-7 cells transfected with miR‑340‑5p mimics. P<0.05 compared with NC mimics group. (D) The predicted binding sites of miR‑340‑5p on 3'UTR of ZEB2 and the target sequences were mutated. (E) Luciferase activity of MCF‑7 cells co‑transfected with ZEB2‑WT or ZEB2‑MUT and miR‑340‑5p mimics or NC mimics. P<0.05 compared with ZEB2‑WT and NC mimics group. (F) The relative expression of miR‑340‑5p in MCF‑7 cells transfected with miR‑340‑5p inhibitors. P<0.05 compared with NC inhibitor group. (G) The protein level of ZEB2 in MCF‑7 cells. miR‑340‑5p mimics repressed ZEB2 expression and miR‑340‑5p inhibitors increased ZEB2 expression. P<0.05. OIP5‑A S1 regulates Z EB2 indirectly through sponging inhibitors. miR‑340‑5p inhibitors alone enhanced invasive miR‑340‑5p. We next explored whether OIP5-AS1 regulates ability. However, knockdown of ZEB2 markedly repressed ZEB2 expression through sponging miR‑340‑5p. The ZEB2 cell invasion even with miR‑340‑5p inhibitors, suggesting that mRNA expression was decreased with either knockdown ZEB2 is a downstream factor (Fig. 4C and D). Overall, these of OIP5‑AS1 or overexpression of miR‑340‑5p mimics; results demonstrated that OIP5-AS1 regulates ZEB2 indirectly however, this effect was reversed by miR‑340‑5p inhibitors through sponging miR‑340‑5p. (Fig. 4A). Then, we tested the protein level of ZEB2. The miR‑340‑5p inhibitors also reversed the repressed effect OIP5‑AS1 promotes breast cancer cells into lung metastasis of OIP5‑AS1 knockdown and miR‑340‑5p inhibitors alone in vivo. To determine whether OIP5-AS1 causes breast cancer upregulated ZEB2 expression. ZEB2 is a known transcrip- cell metastasis in vivo, the metastasis assay was conducted and tional repressor of E-cadherin. In this experiment, we found the primary pulmonary metastasis was observed. We over- that the protein level of E-cadherin was inversely correlated expressed OIP5-AS1 by lentivirus infection in MCF-7 cells with the ZEB2 level (Fig. 4B). Moreover, we examined the cell and then injected cells into nude mice via tail vein (Fig. 5A). invasion ability. The siOIP5-AS1 group showed a decreased The protein level of ZEB2 was elevated by OIP5-AS1 number of invasive cells which was reversed by miR‑340‑5p overexpression (Fig. 5B). The LV-OIP5-AS1 group showed ONCOLOGY REPORTS 44: 1662-1670, 2020 Figure 4. OIP5‑AS1 regulated ZEB2 indirectly through sponging miR‑340‑5p. (A) RT‑qPCR analysis of relative ZEB2 mRNA expression in MCF‑7 cells. * ## ** # Either siOIP5‑AS1 or miR‑340‑5p mimics repressed the ZEB2 expression. P<0.05, P<0.05 vs. siNC; P<0.05, P<0.05 vs. NC mimics. (B) The protein level of ZEB2 and E‑cadherin in MCF‑7 cells transfected with siNC or siOIP5‑AS1 and NC inhibitors or miR‑340‑5p inhibitors. P<0.05. (C) The protein level of ZEB2 in MCF-7 cells transfected with siNC or siZEB2. (D) Transwell invasion assay demonstrated cell invasion in different groups. Less invaded cells were shown with siOIP5‑AS1 transfection but rescued by miR‑340‑5p inhibitor. Figure 5. OIP5-AS1 promoted breast cancer cells into lung metastasis in vivo. (A) RT-qPCR analysis of OIP5-AS1 expression in Lv-vector and Lv-OIP5-AS1 infection cells. P<0.05. (B) The expression of ZEB2 proteins in Lv-vector and Lv-OIP5-AS1 infection cells. (C) The lung tissues of nude mice in Lv-NC and Lv-OIP5-AS1 groups. (D) The number of metastatic lung nodules. P<0.05. (E) H&E staining of metastatic lung nodules in Lv‑NC group and Lv‑OIP5‑AS1 group. (F) Ki‑67 staining of metastatic lung nodules in Lv‑NC group and Lv‑OIP5‑AS1 group. Arrows indicate metastatic nodules. marked lung colonization and increased metastatic lung our observation showing increased metastatic lung nodules nodules compared with the LV-NC group (Fig. 5C and D). (Fig. 5E). Furthermore, the metastatic tumors were positively We performed H&E staining of the metastatic lung tissue stained with Ki‑67 the marker of cell proliferation (Fig. 5F). in the LV-OIP5-AS1 group. The results were consistent with In conclusion, these results demonstrate that overexpression MENG et al: OIP5-AS1 PROMOTES BREAST CANCER METASTASIS VIA miR‑340‑5p/ZEB2 AXIS of OIP5-AS1 promotes breast cancer cells into lung metastasis miR‑340‑5p. Interestingly, it has been reported that miR‑340‑5p in vivo. is negatively associated with distant metastasis in invasive breast cancers, which suggests the pivotal role of miR‑340‑5p in Discussion metastasis (28). Our results elucidate the ability of miR‑340‑5p to target ZEB2 which is a new n fi ding confirmed by regulation In the present study, we investigated the role of long non-coding at both the mRNA and protein levels. Long non-coding RNAs RNA OIP5-AS1 in breast cancer metastasis. We found that have the potential binding ability with multiple miRNAs through OIP5-AS1 was upregulated in five breast cancer cell lines complementary sequences. Several miRNAs were reported which was consistent with earlier studies and in agree- to target OIP5-AS1 in the literature, such as miR-129-5p, ment with supporting evidence from genome-wide analysis miR‑448, miR‑378a‑3p and miR‑498 (21,23,35,36). Notably, of human cancers indicating the prevalent upregulation of in our results, the siOIP5-AS1-suppressed EMT process in OIP5-AS1 (21,29). In vivo experiments also confirmed the breast cancer cells was markedly blocked by miR‑340‑5p effects of OIP5-AS1 in breast cancer cells on lung metas- inhibitors, suggesting a specic fi inhibitory role of miR‑340‑5p tasis. Furthermore, knockdown of OIP5-AS1 markedly for OIP5-AS1 in metastasis process. However, the experiments weakened cell migration and invasion abilities and inhibited validating the OIP5‑AS1/miR‑129‑5p/ZEB2 axis was only epithelial-to-mesenchymal transition (EMT). These results performed in MCF-7 cells which is a potential limitation of suggest the pivotal role of OIP5-AS1 in breast cancer metastasis this study. Therefore, this molecular mechanism needs to be and indicate its potential to be a marker for metastatic breast confirmed in other breast cancer cell lines. Additionally, the cancer or for therapeutic evaluation. Moreover, we provided functions of long non-coding RNA as miRNA sponge allow evidence that ZEB2 is an important effector of OIP5-AS1 us to consider its regulatory networks in tumor biology. More dysregulation and this association was evident through the genome-wide analysis and follow-up functional studies on regulation of miR‑340‑5p. OIP5-AS1 should carried out to understand its diverse role in Emerging evidence reveals the role of long non-coding different types of cancer. RNAs (LncRNAs) in tumorigenesis and tumor metastasis as In conclusion, we identified the OIP5‑AS1/miR‑340‑ the regulator for key gene expression at either transcriptional or 5p/ZEB2 axis in breast cancer cell metastasis. OIP5‑AS1 translational levels (30). Studies interfered metastasis‑associated facilitated breast cancer metastasis by sponging miR‑340‑5p lncRNAs, such as MALAT1, NEAT1 and BCAR4, showed to upregulate ZEB2 mRNA transcripts. The current results signic fi ant metastasis inhibition (14,31,32). OIP5‑AS1 is a newly provide a new direction for the further investigation of identie fi d lncRNA, the dysregulation of which has been found in molecular mechanism of breast cancer metastasis. Defining the multiple cancer types including breast cancer (33). It is involved in underlying mechanisms of differentially expressed lncRNA in cancer cell proliferation showing a G2/M to G0/G1‑phase arrest. cancers may be useful in developing novel strategies for cancer Silencing of OIP5-AS1 has been shown to inhibit cell proliferation diagnosis and treatment. in multiple cancers (20,21,23,24). In addition, downregulation of OIP5-AS1 has been shown to regulate EMT markers E-cadherin Acknowledgements and to reduce metastasis in lung adenocarcinoma (23). Similar results were also obtained in hepatoblastoma demonstrating the Not applicable. involvement of OIP5‑AS1 in EMT progress (34). Together with our n fi dings, the functions of OIP5‑AS1 in cancer metastasis have Funding been verie fi d in multiple cancer types. Thus, further investigations are needed to validate the network of OIP5-AS1 with clinical No funding was received. stages in related cancer types. In our study, we only examined the function of OIP5-AS1 in the regulation of EMT-related proteins Availability of data and materials in MCF‑7 and MDA‑MB‑231 cells. These two breast cancer cell lines represent different molecular subtypes of breast cancer which All data generated or analyzed during the present study are show different metastasis capabilities. Although the regulation included in this published article. of EMT‑related proteins was confirmed in these two cell lines, more experiments should be performed in multiple subtypes of Authors' contributions breast cancer cell lines due to the different metastatic ability and diversity in the molecular interactions involved even in the same LM and HL conceived and designed the study. LM, XY and DZ cancer type. Moreover, the general upregulation of OIP5-AS1 has performed the experiments. LM and HL wrote the manuscript. been revealed in different cell lines, but the varying expression All authors read and approved the final manuscript and agreed values that correlate to metastatic ability is not clear. to be accountable for all aspects of the work in ensuring that OIP5-AS1 was probed using FISH assay was predominantly questions related to the accuracy or integrity of any part of the in the cytoplasm which indicates the potential role of being work are appropriately investigated and resolved. ceRNAs. Findings have shown that lncRNAs act as ceRNAs which compete for miRNAs to regulate the expression of Ethics approval and consent to participate target genes (12). In the present study, we tested miR‑340‑5p according to the predicted binding sequences from TargetScan Animal experiments conducted in the present study were and Starbase tools. 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BioMed Res Int 2015: 320214, 2015. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Oncology Reports Pubmed Central

Long non coding RNA OIP5-AS1 promotes metastasis of breast cancer via miR-340-5p/ZEB2 axis

Oncology Reports , Volume 44 (4) – Aug 10, 2020

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Copyright: © Meng et al.
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1021-335X
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1791-2431
DOI
10.3892/or.2020.7724
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Abstract

ONCOLOGY REPORTS 44: 1662-1670, 2020 Long non coding RNA OIP5‑AS1 promotes metastasis of breast cancer via miR‑340‑5p/ZEB2 axis 1 2 1 3 LINGJUN MENG , XIAOJING YUE , DI ZHOU and HONGJUN LI Department of Hematology and Oncology, China‑Japan Union Hospital Affiliated to Jilin University, Changchun, Jilin 130033; Department of Developmental and Behavioral Pediatrics, The First Hospital of Jilin University, Changchun 130021; Health Management Medical Center, China‑Japan Union Hospital Affiliated to Jilin University, Changchun, Jilin 130033, P.R. China Received January 22, 2020; Accepted July 20, 2020 DOI: 10.3892/or.2020.7724 Abstract. Breast cancer is the most common invasive cancer Introduction in women with the highest number of related deaths which is caused by distal metastasis. Recently, integrated analysis of Currently, breast cancer remains a leading health problem and gene expression profile suggested widespread gene dysregula - constitutes one of the most severe burdensome diseases in tion in various types of cancer. Research in the past decade females around the world despite understanding of underlying has focused on long non-coding RNAs (lncRNAs), particu- molecular mechanisms (1). Tumor metastasis is diagnosed larly in cell proliferation, tumor progression and metastasis. in approximately 30% of breast cancer patients and is the OPA-interacting protein 5 antisense transcript 1 (OIP5-AS1) major cause of cancer-related deaths (2). The prognosis for is an evolutionarily conserved long non-coding RNA that most patients with metastatic breast cancer is unfavorable has been linked to oncogenesis in multiple cancers. In breast with a median overall survival range from 2 to 3 years (3). cancer, dysregulation of OIP5-AS1 was reported but the Generally, breast cancer can be categorized into four subtypes precise role in cancer development and progression remains [luminal A, luminal B, human epidermal growth factor unclear. In the present study, using small interfering RNA receptor 2 (HER2) positive and triple negative], which are (siRNA) targeting OIP5-AS1, it was shown that knockdown defined using immunohistochemical breast tumor markers (4). of OIP5-AS1 was associated with alteration of EMT markers These four subtypes have the potential risk of distant metas- and suppressed migration and invasion of breast cancer tasis but with differential site‑specic fi metastatic patterns (5). cells. Among the EMT-related transcription factors, ZEB1 Currently, breast cancer metastasis from primary tumor to and ZEB2 were signic fi antly downregulated with OIP5‑AS1 distant organs occurs through a sequential molecular cascade knockdown. Computational analysis and a dual-luciferase including local angiogenesis for tumor growth, invasion of the reporter system identified miR‑340‑5p was the target gene surrounding tissue, intravasation of the carcinoma cells into for OIP5‑AS1. Further experiments verified the function of the blood or lymphatic vessels, dissemination and proliferation OIP5‑AS1 in cell invasion was dependent on miR‑340a‑5p at secondary neoplastic foci (6). These carcinoma cells obtain through regulating target gene ZEB2. In vivo study demon- mesenchymal features and suppress their epithelial features strated that overexpressing OIP5-AS1 in breast cancer cells through the epithelial-to-mesenchymal transition (EMT) promoted lung metastasis in nude mice. The findings of the process to promote an invasive and metastatic phenotype (7). present study revealed the mechanism of OIP5-AS1 in breast Multiple transcription factors coordinate EMT programs. cancer metastasis. Overall, our study may provide a potential Among them, zinc‑finger E‑box‑binding (ZEB) transcription therapeutic target for breast cancer metastasis. factors, ZEB1 and ZEB2, are two EMT regulators that either repress or activate transcription in various types of cancer (8). Furthermore, ZEB2 was reported to negatively correlate with the epithelial marker E-cadherin in breast cancer cells involved in breast cancer progression (9). Long non-coding RNAs (lncRNAs) are a class of tran- Correspondence to: Professor Hongjun Li, Health Management scripts containing more than 200 nucleotides in length with Medical Center, China-Japan Union Hospital Affiliated to limited protein‑coding capacity (10). Recent findings have Jilin University, 126 Xiantai Street, Changchun, Jilin 130033, shown that dysregulation of lncRNAs is involved in cell prolif- P.R. China eration, tumor progression and metastasis in cancers (11). E-mail: hj_li@jlu.edu.cn Functionally, lncRNAs interact with proteins and other RNAs Key words: OIP5‑AS1, miR‑340‑5p, ZEB2, breast cancer, to regulate their activities and cellular location. Furthermore, metastasis lncRNAs act as molecular sponges for miRNAs that block the binding activity for target transcripts (12). In breast cancer, several lncRNAs have been identified as either oncogenic MENG et al: OIP5-AS1 PROMOTES BREAST CANCER METASTASIS VIA miR‑340‑5p/ZEB2 AXIS or tumor suppressive factors, such as X-inactive-specific- non-coated chambers with a pore size of 0.8 µM. The trans- transcript (XIST), HOX antisense intergenic RNA (HOTAIR), fected cells were seeded into the upper chamber in DMEM growth arrest specic fi 5 (GAS5) and metastasis‑associated lung with 1% FBS and the lower chamber was filled with 10% FBS adenocarcinoma transcript 1 (MALAT1) (13‑16). A systematic as a chemoattractant. After 24‑h incubation in the humidie fi d analysis of the correlation has been carried out between these incubator at 37˚C with 5% CO , cells in the upper chamber dysregulated lncRNAs and breast cancer clinicopathology and were removed and cells in the lower side were fixed with survival suggesting a pivotal role in cancer development (17). 4% PFA and stained by 1% crystal violet. Stained cells were Increasing lncRNAs have been shown to participate in specic fi then visualized and imaged at a x20 magnic fi ation by a light cancer types but more often exert general function in a broad microscope. spectrum of cancer. OPA-interacting protein 5 antisense transcript 1 (OIP5-AS1) Western blot analysis. The cellular proteins were extracted is an evolutionarily conserved long non-coding RNA that is in RIPA buffer supplemented with protease inhibitor. The transcribed from opposite direction to the OIP5 gene. It was protein concentration was quantie fi d using BCA protein assay first shown to be expressed in the nervous system and was and 20 µg of each protein sample was loaded and analyzed by essential for neurogenesis during embryonic development (18). 10% sodium dodecyl sulfate (SDS)‑polyacrylamide gel elec- The functions of OIP5-AS1 in multiple human cancers have trophoresis (PAGE) system. Then, proteins were transferred to been reported to be associated with oncogenesis (19,20). a PVDF membrane. The membrane was blocked in 5% BSA In breast cancer, OIP5-AS1 levels are upregulated in breast and probed with primary antibodies: Anti‑ZEB2 (1:1,000; tumor tissue and correlated with tumor size, metastatic status no. ab138222, Abcam), anti‑E‑cadherin (1:1,000; no. 14472, of lymph nodes, pathological grading and TNM stage (21). Cell signaling), anti‑N‑cadherin (1:1,000; no. ab18203, Abcam), In the present study, we investigated the role of OIP5-AS1 anti‑vimentin (1:1,000; no. ab92547, Abcam), anti‑ZEB1 in breast cancer metastasis using the in vitro and in vivo (1:1,000; no. 70512, Cell signaling), anti‑Snail (1:1,000; models showing that OIP5-AS1 regulates ZEB2 expression by no. IMG‑6639A, Novus Biologicals), anti‑Slug (1:1,000; acting as ceRNA for miR‑340‑5p. no. 9585, Cell signaling), anti‑Twist (1:1,000; no. 69366, Cell signaling) and anti‑ GA PDH (1:2,0 0 0; no. ab8245, Materials and methods Abcam). Then, the membrane was incubated with peroxi- dase-conjugated anti-mouse or anti-rabbit secondary antibody Cell culture and transfection. Breast cancer cell lines MCF-7, (1:2,000, nos. NEF822001EA; NEF812001EA, PerkinElmer). MDA‑MB‑231, ZR‑75, MDA‑MB‑468, SKBR3 and normal Immunoreactivity bands were detected by chemiluminescence human epithelial cell line MCF-10A were purchased from the and the intensity of the bands was quantie fi d using Image Lab American Type Culture Collection (ATCC). MCF-10A cells Software (Bio Rad, China). were cultured in MEBM (Lonza) and supplemented with 100 ng/ml cholera toxin. ZR‑75 and SKBR3 cell lines were RNA fluorescence in situ hybridization (FISH). A Cy3‑labeled maintained in RPMI‑1640 medium (Sigma) containing 10% set of probes recognizing OIP5-AS1 was designed and synthe- FBS, 2 mM L‑glutamine and 2% penicillin and streptomycin. sized by Biosearch Technologies. The MCF-7 cells were MCF‑7, MDA‑MB‑231, and MDA‑MB‑468 cell lines were cultured on coverslips for 24 h and then fixed in 4% PFA. After maintained in DMEM supplemented with 10% FBS plus permeabilization with 70% ethanol at 4˚C for 1 h, cells were 2% penicillin and streptomycin. Cells were cultured in a hybridized with the OIP5-AS1 probes dissolved in hybridiza- humidie fi d incubator at 37˚C with 5% CO . tion buffer (no. SMF-HB1-10, Biosearch Technologies) at The pre-designed siOIP5-AS1 and siZEB2 were 37˚C in the dark for 16 h. The nucleus was stained with DAPI. purchased from ThermoFisher (no. 4390771, no. AM16708) Images were captured using a confocal microscope (Olympus). and transfected into cells using Lipofectamine RNAiMAX reagent (ThermoFisher) following the manufacturer's Quantitative real‑time PCR (RT‑PCR). Total RNA was instructions. miR‑340‑5p mimics, negative control mimics, ext r a ct e d f rom c el ls ( MC F‑7, M DA‑M B ‑231, Z R‑75, miR‑340‑5p inhibitors and negative control inhibitors were MDA‑MB‑468, SKBR3, and MCF‑10A) using TRIzol reagent purchased from GenePharma and transfected into cells using (Thermo Fisher). Total RNA (1 µg) was reverse transcribed Lipofectamine 2000 reagent (ThermoFisher), according to the into cDNA and a SYBR-Green quantitative real-time PCR manufacturers' instructions. miR‑340‑5p mimic: 5'‑UUA UAA Master Mix kit was used to detect qPCR signals. The targeted AGC AAU GAG ACU GAU U‑3' and miR‑340‑5p inhibitor: gene expression was normalized with GAPDH and calcu- - Cq ΔΔ 5'‑AAU CAG UCU CAU UGC UUU AUA A‑3'. Cells were used lated using 2 method (22). The primer sequences used for further experiments at 48 h after transfection. were: OIP5‑AS1: 5'‑TGC AAC CCA AGG TGG ATA CT‑3' and 5'‑GAG AGA CTG CAG TGA GCA GA‑3'; ZEB2: 5'‑CAG CTC Wound healing assay. MCF‑7 and MDA‑MB‑231 cells were TTC CAC CTC AAA GC‑3' and 5'‑TCC TTG TTT CCG CTG seeded in 12-well plates and transfected with either siNC or GTA CT‑3'; GAPDH: 5'‑GT C GGA GTC AAC GGA TTT GG‑3' siOIP5-AS1. A linear wound was scratched across the center of and 5'‑TGA CGG TGC CAT GGA ATT TG‑3'. For the detection the well using a sterile pipette tip. The images of wound closure of miR‑340‑5p, stem‑loop qRT‑PCR was performed using were captured after 24 h using Olympus microscope (x10). miScript SYBR‑Green PCR Kit with U6 small nuclear RNA as an internal control (Qiagen). The following thermocycling Transwell invasion assay. The invasion of MCF-7 and conditions were used in the experiments: PCR initial activa- MDA‑MB‑231 cells was detected using matrigel‑coated or tion at 95˚C for 15 min, followed by 40 cycles of denaturation ONCOLOGY REPORTS 44: 1662-1670, 2020 at 94˚C for 15 sec, annealing at 55˚C for 30 sec and an exten- with LV-OIP5-AS1 or LV-NC were intravenously injected sion at 70˚C for 30 sec. through the tail vein of BALB/c nude mice under isou fl rane anaesthesia. After 8 weeks of inoculation, the mice were RNA immunoprecipitation assay. Magna RIP kit (Millipore) euthanized and the number of lung metastatic tumors per lung was used for RNA immunoprecipitation experiments. The were counted under a dissecting microscope and confirmed by procedure was performed following the manufacturer's H&E staining. The experimental protocols were approved by protocol. Brief ly, after miR‑340‑5p mimics or NC mimics the Animal Care Committee of China-Japan Union Hospital transfection, the cells were lysed in RIP lysis buffer. The cell Affiliated to Jilin University. lysate was incubated with either Ago2 antibody or control IgG together with protein A/G magnetic beads. Then the beads Statistical analysis. Data are expressed as mean ± standard were washed and incubated with Proteinase K at 55˚C for error mean (SEM). The Student's t test was employed to 30 min to digest proteins. The purie fi d RNA was obtained and compare two groups and one-way ANOVA with post hoc analyzed by RT-qPCR. test was used to analyze differences among multiple groups. A value of P<0.05 was considered as statistically signic fi ant. Dual‑luciferase reporter assay. In this study, the OI P5 ‑AS1/m i R NA i nt er a ct ions were pre d ict e d usi ng Results Starbase (http://starbase.sysu.edu.cn/) and DIANA‑LncBase database (http://www.microrna.gr/LncBase). For OIP5‑AS1 Interference of OIP5‑AS1 represses epithelial‑to‑mesenchymal a nd m i R‑34 0 ‑5p binding activit y, OI P5‑AS1 f ragment transition (EMT) in breast cancer cells by regulating ZEB containing the binding sites of miR‑340‑5p, as well as those family proteins. The dysregulation of long non-coding RNA of the wild-type and mutant sequences were cloned into a OIP5-AS1 was involved in multiple cancer types associating pmirGLO Dual-luciferase Vector designated as OIP5-AS1 with overall survival, TNM stage and prognosis (21,23‑25). In WT or OIP5‑AS1 MUT. For ZEB2 and miR‑340‑5p binding breast cancer, studies reported that OIP5-AS1 is upregulated activity, fragment of 3'UTR ZEB2 containing the binding in both tumor samples and cell lines (21). We first evaluated sites of miR‑340‑5p, as well as the wild‑type and mutant the expression levels of OIP5‑AS1 in v fi e breast cancer cell sequences were cloned into a pmirGLO Dual-luciferase lines. The results showed that the relative expression levels of Vector designated as ZEB2 WT or ZEB2 MUT. These vectors OIP5‑AS1 were much higher in the v fi e breast cancer cell lines were co‑transfected with either NC mimics or miR‑340‑5p than in the normal epithelial cell line MCF-10A (Fig. 1A). mimics using Lipofectamine 2000 reagent. At 48 h after Then, we chose luminal-type breast cancer cell line MCF-7 transfection, the relative luciferase activities were recorded and basal B TNBC cell line MDA‑MB‑231 for further func- by dual- luciferase reporter assay system (Promega) and the tional studies. To investigate the role of OIP5-AS1 in breast values were normalized to the Renilla luciferase activity. cancer metastasis, we efficiently knocked down OIP5‑AS1 with siRNAs in MCF‑7 and MDA‑MB‑231 cell lines (Fig. 1B) Immunohistochemistry staining and hematoxylin and eosin and analyzed the cell migration and invasion properties. In (H&E) staining. The lung of nude mice was dissected and the wound healing assay, siOIP5-AS1 groups showed a slower fixed in 10% formaldehyde at room temperature overnight. migration rate than the siNC group (Fig. 1C). Furthermore, The embedded samples in paraffin were sectioned into 5 µm knockdown of OIP5‑AS1 in the two cell lines significantly slices and mounted on glass slides. For immunohistochemical inhibited cell invasion (Fig. 1D). Next, we assessed the effects staining, the slides of interest were probed with anti‑Ki‑67 of the downregulation of OIP5-AS1 on the expression of antibodies (1:500; Abcam) and then the secondary streptav- epithelial-to-mesenchymal transition (EMT) markers. The idin-horseradish peroxidase-conjugated antibody staining. protein analysis results indicated that the epithelial marker Immunoreactivity was visualized by DAB and lightly E-cadherin was increased whereas the mesenchymal markers counterstained with 5% hematoxylin. For H&E staining, N-cadherin and Vimentin were decreased (Fig. 1E). These slides were deparaffinized and rehydrated in graded ethanol results suggested that downregulation of OIP5-AS1 repressed solutions, then in distilled water. After H&E staining, slides epithelial-to-mesenchymal transition (EMT). Considering the were mounted and examined under a light microscope. importance of transcription factors in EMT co-ordination, we further tested the expression of EMT-related transcrip- Lentivirus production and in vivo metastasis assay. Full-length tion factors (ZEB1, ZEB2, Snail, Slug and Twist). In the cDNA of human OIP5‑AS1 was amplie fi d from the mRNA of siOIP5‑AS1 group, ZEB1 and ZEB2 were signic fi antly down - MCF‑7 cells and subcloned into pcDNA3.1 (AddGene). The regulated whereas the expression of Snail, Slug and Twist were lentiviral and packaging vectors (AddGene) were co-transfected not affected (Fig. 1F). Thus, we speculated that OIP5-AS1 into HEK293FT cells using Lipofectamine 2000 reagent may exert functions in EMT through regulating ZEB family (ThermoFisher) according to the manufacturer's instructions. proteins. Virus was collected and concentrated at 48 h after transfection. Twenty healthy 6‑ to 8‑week‑old female BALB/c nude mice OIP5‑AS1 directly targeted miR‑340 ‑5p. LncRNAs exert (The Animal Institute, Jilin University) were used in this study function in various aspects of cellular function and biological and randomly divided into two groups. The mice were housed process in either nucleus or cytoplasm. In nucleus, lncRNAs in a specific pathogen‑free (SPF) facility and exposed to a may take part in chromatin remodeling and modic fi ation or 12‑h light/dark cycle. Water and food were offered ad libitum. gene expression prior to transcription, whereas lncRNAs After 1 week of acclimatization, MCF-7 cells (1x10 ) infected in cytoplasm mainly participate in post-transcriptional MENG et al: OIP5-AS1 PROMOTES BREAST CANCER METASTASIS VIA miR‑340‑5p/ZEB2 AXIS Figure 1. Downregulation of OIP5-AS1 repressed migration, invasion and epithelial-to-mesenchymal transition in breast cancer cells. (A) RT-qPCR anal- ysis of OIP5-AS1 expression in breast cancer cell lines compared with normal mammary MCF-10A epithelial cells. P<0.05. (B) RT-qPCR analysis of ** OIP5‑AS1 expression with siNC or siOIP5‑AS1 transfection in MCF‑7 and MDA‑MB‑231 cells. P<0.01 compared with siNC. (C) Migration of MCF-7 and MDA‑MB‑231 cells in siNC or siOIP5‑AS1 transfection groups was determined by wound healing assay. siOIP5‑AS1 resulted at a slower closing rate. (D) Transwell invasion assay demonstrated less invaded cells with siOIP5‑AS1 transfection in MCF‑7 and MDA‑MB‑231 cells. (E) The protein expression of EMT markers (E-cadherin, N-cadherin and vimentin) indicated the suppression of epithelial-to-mesenchymal transition with siOIP5-AS1 transfection in MCF‑7 and MDA‑MB‑231 cells. GAPDH was used as an internal control. (F) The protein level of EMT‑related transcriptional factors (ZEB2, ZEB1, Snail, Slug and Twist) in siNC or siOIP5-AS1 transfection groups indicating the selective regulation of ZEB2 and ZEB1. GAPDH was used as an internal control. P<0.05. NC, negative control. regulation and post‑translational modic fi ation (26,27). Thus, was mainly localized in the cytoplasm in MCF-7 cells which we assessed subcellular location of OIP5-AS1 by fluores- indicated OIP5-AS1 may serve as a ceRNA in breast cancer cence in situ hybridization (FISH). The detected OIP5-AS1 cells (Fig. 2A). ONCOLOGY REPORTS 44: 1662-1670, 2020 Figure 2. OIP5‑AS1 directly targeted miR340‑5p. (A) RNA FISH probed endogenous OIP5‑AS1 (red) was located in cytoplasm in MCF‑7 cells. Scale bar, 10 µm. (B) RNA‑IP analysis confirmed the direct binding of miR‑340‑5p mimics and OIP5‑AS1 in MCF‑7 cells. The expression of OIP5‑AS1 was detected using RT-qPCR. P<0.05. (C) The predicted binding sites of miR‑340‑5p on OIP5‑AS1, and target sequences were mutated. (D) Luciferase activity of MCF‑7 ** cells co‑transfected with OIP5‑AS1 WT or OIP5‑AS1 MUT and miR‑340‑5p mimics or NC mimics. P<0.01 compared with OIP5-AS1 WT and NC mimics ** group. (E) Stem‑loop RT‑qPCR analysis of miR‑340‑5p with siNC or siOIP5‑AS1 transfection in MCF‑7 and MDA‑MB‑231 cells. P<0.01 compared with siNC group. In this study, the OIP5‑AS1/miRNA interactions were expression of miR‑340‑5p in breast cancer cell lines. The predicted using Starbase and DIANA tools. Among the level of miR‑340‑5p was decreased in MCF‑7, MDA‑MB‑231, predicted miRNAs, miR‑340‑5p possesses three target sites ZR‑75, MDA‑MB‑ 468 and SKBR3 cells as compared to on OIP5‑AS1. In order to confirm that miR‑340‑5p is the human breast epithelial cell line MCF10A (Fig. 3A). target gene of OIP5-AS1, we per for med anti-Ago2 RIP assay Next, we screened mRNA targets of miR‑340‑5p using and dual luciferase reporter assay. In anti-Ago2 RIP assay, TargetScan and Starbase tools and found the 3'UTR of the endogenous OIP5-AS1 was specifically enriched in ZEB2 mRNA contains two binding sites for miR‑340‑5p. miR‑340‑5p mimics‑transfected cells when compared with We transfected miR‑340‑5p mimics into MCF‑7 cells and NC mimics group (Fig. 2B). We constructed the OIP5-AS1 detected the expression of ZEB2 mRNA by RT-qPCR. With wild-type and mutant reporter plasmids according to the the miR‑340‑5p overexpression (Fig. 3B), the level of ZEB2 bi ndi ng sequences of m i R‑34 0 ‑5p ( Fig. 2C). T he dua l m R NA was decreased (Fig. 3C). We fur ther confir med luciferase reporter assay showed that the reduced lucif- the direct binding between ZEB2 mRNA and miR‑340‑5p erase activity was only found in the miR‑340‑5p mimics by dual luciferase reporter assay. The ZEB2 3'UTR was and OIP5-AS1 wild-type co-transfection groups but not constructed and the mutant form was designed according to in the OIP5-AS1 mutant co-transfection group (Fig. 2D). the miR‑340‑5p binding sequences (Fig. 3D). As shown in Moreover, we tested the expression of m iR‑340 ‑5p with Fig. 3E, the luciferase activity was only reduced in the ZEB2 OIP5-AS1 knockdown in breast cancer cells. After two 3'UTR wild‑type and miR‑340‑5p mimics co‑transfection days with siOIP5‑AS1 transfection, the level of miR‑340‑5p group which suggested the direct binding between ZEB2 was increased in MCF‑7 and MDA‑MB‑231 cells (Fig. 2E). m R NA and m iR‑340 ‑5p. In addition, we exam ined the Taken together, these results confirmed the direct binding effects of miR‑340‑5p on the protein expression of ZEB2 by act ivit y bet we en OI P5 ‑AS1 a nd m i R‑34 0 ‑5p i n breast overexpression of either miR‑340‑5p mimics or inhibitors in cancer cells. MCF-7 cells. Similarly, the level of ZEB2 was decreased with miR‑340‑5p mimics transfection whereas it was increased miR‑340 ‑5p is downregulated in breast cancer cells and with miR‑340‑5p inhibitors transfection (Fig. 3F and G). regulates ZEB2 expression. A recent study reported that Collectively, these results supported that miR‑340‑5p regulates miR‑340‑5p was negatively associated with distant metastasis ZEB2 expression by binding to complementary sequences in in invasive breast cancers (28). Thus, we measured the relative the 3'UTR of ZEB2 mRNA. MENG et al: OIP5-AS1 PROMOTES BREAST CANCER METASTASIS VIA miR‑340‑5p/ZEB2 AXIS Figure 3. miR‑340‑5p is downregulated in breast cancer cells and regulates ZEB2 expression. (A) RT‑qPCR analysis of miR‑340‑5p expression in breast cancer cell lines compared with normal mammary MCF-10A epithelial cells. P<0.05. (B) The relative expression of miR‑340‑5p in MCF‑7 cells transfected with ** miR‑340‑5p mimics. P<0.01 compared with NC mimics group. (C) RT-qPCR analysis of relative ZEB2 mRNA expression in MCF-7 cells transfected with miR‑340‑5p mimics. P<0.05 compared with NC mimics group. (D) The predicted binding sites of miR‑340‑5p on 3'UTR of ZEB2 and the target sequences were mutated. (E) Luciferase activity of MCF‑7 cells co‑transfected with ZEB2‑WT or ZEB2‑MUT and miR‑340‑5p mimics or NC mimics. P<0.05 compared with ZEB2‑WT and NC mimics group. (F) The relative expression of miR‑340‑5p in MCF‑7 cells transfected with miR‑340‑5p inhibitors. P<0.05 compared with NC inhibitor group. (G) The protein level of ZEB2 in MCF‑7 cells. miR‑340‑5p mimics repressed ZEB2 expression and miR‑340‑5p inhibitors increased ZEB2 expression. P<0.05. OIP5‑A S1 regulates Z EB2 indirectly through sponging inhibitors. miR‑340‑5p inhibitors alone enhanced invasive miR‑340‑5p. We next explored whether OIP5-AS1 regulates ability. However, knockdown of ZEB2 markedly repressed ZEB2 expression through sponging miR‑340‑5p. The ZEB2 cell invasion even with miR‑340‑5p inhibitors, suggesting that mRNA expression was decreased with either knockdown ZEB2 is a downstream factor (Fig. 4C and D). Overall, these of OIP5‑AS1 or overexpression of miR‑340‑5p mimics; results demonstrated that OIP5-AS1 regulates ZEB2 indirectly however, this effect was reversed by miR‑340‑5p inhibitors through sponging miR‑340‑5p. (Fig. 4A). Then, we tested the protein level of ZEB2. The miR‑340‑5p inhibitors also reversed the repressed effect OIP5‑AS1 promotes breast cancer cells into lung metastasis of OIP5‑AS1 knockdown and miR‑340‑5p inhibitors alone in vivo. To determine whether OIP5-AS1 causes breast cancer upregulated ZEB2 expression. ZEB2 is a known transcrip- cell metastasis in vivo, the metastasis assay was conducted and tional repressor of E-cadherin. In this experiment, we found the primary pulmonary metastasis was observed. We over- that the protein level of E-cadherin was inversely correlated expressed OIP5-AS1 by lentivirus infection in MCF-7 cells with the ZEB2 level (Fig. 4B). Moreover, we examined the cell and then injected cells into nude mice via tail vein (Fig. 5A). invasion ability. The siOIP5-AS1 group showed a decreased The protein level of ZEB2 was elevated by OIP5-AS1 number of invasive cells which was reversed by miR‑340‑5p overexpression (Fig. 5B). The LV-OIP5-AS1 group showed ONCOLOGY REPORTS 44: 1662-1670, 2020 Figure 4. OIP5‑AS1 regulated ZEB2 indirectly through sponging miR‑340‑5p. (A) RT‑qPCR analysis of relative ZEB2 mRNA expression in MCF‑7 cells. * ## ** # Either siOIP5‑AS1 or miR‑340‑5p mimics repressed the ZEB2 expression. P<0.05, P<0.05 vs. siNC; P<0.05, P<0.05 vs. NC mimics. (B) The protein level of ZEB2 and E‑cadherin in MCF‑7 cells transfected with siNC or siOIP5‑AS1 and NC inhibitors or miR‑340‑5p inhibitors. P<0.05. (C) The protein level of ZEB2 in MCF-7 cells transfected with siNC or siZEB2. (D) Transwell invasion assay demonstrated cell invasion in different groups. Less invaded cells were shown with siOIP5‑AS1 transfection but rescued by miR‑340‑5p inhibitor. Figure 5. OIP5-AS1 promoted breast cancer cells into lung metastasis in vivo. (A) RT-qPCR analysis of OIP5-AS1 expression in Lv-vector and Lv-OIP5-AS1 infection cells. P<0.05. (B) The expression of ZEB2 proteins in Lv-vector and Lv-OIP5-AS1 infection cells. (C) The lung tissues of nude mice in Lv-NC and Lv-OIP5-AS1 groups. (D) The number of metastatic lung nodules. P<0.05. (E) H&E staining of metastatic lung nodules in Lv‑NC group and Lv‑OIP5‑AS1 group. (F) Ki‑67 staining of metastatic lung nodules in Lv‑NC group and Lv‑OIP5‑AS1 group. Arrows indicate metastatic nodules. marked lung colonization and increased metastatic lung our observation showing increased metastatic lung nodules nodules compared with the LV-NC group (Fig. 5C and D). (Fig. 5E). Furthermore, the metastatic tumors were positively We performed H&E staining of the metastatic lung tissue stained with Ki‑67 the marker of cell proliferation (Fig. 5F). in the LV-OIP5-AS1 group. The results were consistent with In conclusion, these results demonstrate that overexpression MENG et al: OIP5-AS1 PROMOTES BREAST CANCER METASTASIS VIA miR‑340‑5p/ZEB2 AXIS of OIP5-AS1 promotes breast cancer cells into lung metastasis miR‑340‑5p. Interestingly, it has been reported that miR‑340‑5p in vivo. is negatively associated with distant metastasis in invasive breast cancers, which suggests the pivotal role of miR‑340‑5p in Discussion metastasis (28). Our results elucidate the ability of miR‑340‑5p to target ZEB2 which is a new n fi ding confirmed by regulation In the present study, we investigated the role of long non-coding at both the mRNA and protein levels. Long non-coding RNAs RNA OIP5-AS1 in breast cancer metastasis. We found that have the potential binding ability with multiple miRNAs through OIP5-AS1 was upregulated in five breast cancer cell lines complementary sequences. Several miRNAs were reported which was consistent with earlier studies and in agree- to target OIP5-AS1 in the literature, such as miR-129-5p, ment with supporting evidence from genome-wide analysis miR‑448, miR‑378a‑3p and miR‑498 (21,23,35,36). Notably, of human cancers indicating the prevalent upregulation of in our results, the siOIP5-AS1-suppressed EMT process in OIP5-AS1 (21,29). In vivo experiments also confirmed the breast cancer cells was markedly blocked by miR‑340‑5p effects of OIP5-AS1 in breast cancer cells on lung metas- inhibitors, suggesting a specic fi inhibitory role of miR‑340‑5p tasis. Furthermore, knockdown of OIP5-AS1 markedly for OIP5-AS1 in metastasis process. However, the experiments weakened cell migration and invasion abilities and inhibited validating the OIP5‑AS1/miR‑129‑5p/ZEB2 axis was only epithelial-to-mesenchymal transition (EMT). These results performed in MCF-7 cells which is a potential limitation of suggest the pivotal role of OIP5-AS1 in breast cancer metastasis this study. Therefore, this molecular mechanism needs to be and indicate its potential to be a marker for metastatic breast confirmed in other breast cancer cell lines. Additionally, the cancer or for therapeutic evaluation. Moreover, we provided functions of long non-coding RNA as miRNA sponge allow evidence that ZEB2 is an important effector of OIP5-AS1 us to consider its regulatory networks in tumor biology. More dysregulation and this association was evident through the genome-wide analysis and follow-up functional studies on regulation of miR‑340‑5p. OIP5-AS1 should carried out to understand its diverse role in Emerging evidence reveals the role of long non-coding different types of cancer. RNAs (LncRNAs) in tumorigenesis and tumor metastasis as In conclusion, we identified the OIP5‑AS1/miR‑340‑ the regulator for key gene expression at either transcriptional or 5p/ZEB2 axis in breast cancer cell metastasis. OIP5‑AS1 translational levels (30). Studies interfered metastasis‑associated facilitated breast cancer metastasis by sponging miR‑340‑5p lncRNAs, such as MALAT1, NEAT1 and BCAR4, showed to upregulate ZEB2 mRNA transcripts. The current results signic fi ant metastasis inhibition (14,31,32). OIP5‑AS1 is a newly provide a new direction for the further investigation of identie fi d lncRNA, the dysregulation of which has been found in molecular mechanism of breast cancer metastasis. Defining the multiple cancer types including breast cancer (33). It is involved in underlying mechanisms of differentially expressed lncRNA in cancer cell proliferation showing a G2/M to G0/G1‑phase arrest. cancers may be useful in developing novel strategies for cancer Silencing of OIP5-AS1 has been shown to inhibit cell proliferation diagnosis and treatment. in multiple cancers (20,21,23,24). In addition, downregulation of OIP5-AS1 has been shown to regulate EMT markers E-cadherin Acknowledgements and to reduce metastasis in lung adenocarcinoma (23). Similar results were also obtained in hepatoblastoma demonstrating the Not applicable. involvement of OIP5‑AS1 in EMT progress (34). Together with our n fi dings, the functions of OIP5‑AS1 in cancer metastasis have Funding been verie fi d in multiple cancer types. Thus, further investigations are needed to validate the network of OIP5-AS1 with clinical No funding was received. stages in related cancer types. In our study, we only examined the function of OIP5-AS1 in the regulation of EMT-related proteins Availability of data and materials in MCF‑7 and MDA‑MB‑231 cells. These two breast cancer cell lines represent different molecular subtypes of breast cancer which All data generated or analyzed during the present study are show different metastasis capabilities. Although the regulation included in this published article. of EMT‑related proteins was confirmed in these two cell lines, more experiments should be performed in multiple subtypes of Authors' contributions breast cancer cell lines due to the different metastatic ability and diversity in the molecular interactions involved even in the same LM and HL conceived and designed the study. LM, XY and DZ cancer type. Moreover, the general upregulation of OIP5-AS1 has performed the experiments. LM and HL wrote the manuscript. been revealed in different cell lines, but the varying expression All authors read and approved the final manuscript and agreed values that correlate to metastatic ability is not clear. to be accountable for all aspects of the work in ensuring that OIP5-AS1 was probed using FISH assay was predominantly questions related to the accuracy or integrity of any part of the in the cytoplasm which indicates the potential role of being work are appropriately investigated and resolved. ceRNAs. Findings have shown that lncRNAs act as ceRNAs which compete for miRNAs to regulate the expression of Ethics approval and consent to participate target genes (12). In the present study, we tested miR‑340‑5p according to the predicted binding sequences from TargetScan Animal experiments conducted in the present study were and Starbase tools. 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Oncology ReportsPubmed Central

Published: Aug 10, 2020

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