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CircMMP11 regulates proliferation, migration, invasion, and apoptosis of breast cancer cells through miR-625-5p/ZEB2 axis

CircMMP11 regulates proliferation, migration, invasion, and apoptosis of breast cancer cells... Background: Circular RNAs (circRNAs) have been demonstrated to play significant roles in regulating gene expres- sion in tumorigenesis, including breast cancer (BC). This study was designed to explore the role and underlying molecular mechanisms of circMMP11 in BC. Methods: The real-time quantitative polymerase chain reaction (RT-qPCR) assay was used for examining expression of circMMP11, microRNA-625-5p (miR-625-5p), and Zinc finger E-box binding homeobox-2 (ZEB2). The protein expres- sion of ZEB2, Vimentin, and E-cadherin was assessed by western blot assay. The proliferation ability of BC cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT ) and colony-forming assays. The transwell assay was used to measure migration and invasion of BC cells. The apoptotic cells were examined by flow cytometry assay. The interaction association among circMMP11, miR-625-5p, and ZEB2 was confirmed by RNA pull-down and dual-luciferase report assays. A xenograft experiment was established to clarify the role of circMMP11 silencing in vivo. Results: We found that circMMP11 and ZEB2 were overexpressed in BC tissues and cells compared with controls. The suppression of circMMP11 or ZEB2 repressed proliferation, migration, and invasion while induced apoptosis of BC cells. Additionally, miR-625-5p, interacted with ZEB2, was a target of circMMP11 in BC cells. CircMMP11 regulated the expression of ZEB2 by targeting miR-625-5p. Knockdown of circMMP11-mediated effects on BC cells could be abol- ished by overexpression of ZEB2. Consistently, silencing of circMMP11 impeded the tumor growth in vivo. Conclusions: CircMMP11/miR-625-5p/ZEB2 axis affected proliferation, migration, invasion, and apoptosis of BC cells through the mechanism of competing endogenous RNAs (ceRNA), indicating that circMMP11 was an oncogenic circRNA in BC. Keywords: circRNA, circMMP11, miR-625-5p, ZEB2, BC Introduction the statistic [2]. Although improved prognosis of patients Breast cancer (BC) is a common female cancer all over with BC had achieved, BC remains leading reason of the world [1]. The morbidity of BC has been rising, over cancer mortality among females [3]. Therefore, it is 2  million females were diagnosed with BC according to meaningful to look into pathogenesis and find diagnosis biomarkers of BC. Circular RNAs (circRNAs), a group of RNA molecules *Correspondence: uncsuj@163.com 1 with closed covalent loops, play critical roles in initia- Department of Breast Surgical Oncology, Cancer Institute and Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union tion and development of the multiple malignancies [4]. Medical College, No. 17 Panjiayuan Nanli, Chaoyang District, Accumulating evidence has revealed multiple charac- Beijing 100021, China terization of circRNAs, including microRNA sponges Full list of author information is available at the end of the article © The Author(s) 2021. 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://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Qi et al. Cancer Cell Int (2021) 21:133 Page 2 of 12 [5], CircMMP11 (hsa_circ_0062558) is derived from the presented in Table  1. BC patients were selected for fur- Matrix Metalloprotease 11 (MMP11) gene and located ther investigation based on two strict criteria: (1) patients on chr22 (24,125,597–24,126,503). A previous research were first diagnosed with BC; (2) patients provided the reported that a series of differentially expressed circR - written informed consent. Exclusion criteria: (1) patients NAs could function as novel biomarkers for BC, includ- received neoadjuvant therapy; (2) patients with other ing circMMP11 [6]. Interestingly, circMMP11 could complicated diseases. function as a competitive endogenous RNA (ceRNA) for miR-1204 to participate in the development of BC, indi- Cell lines and cell culture cating that circMMP11 was a potential therapeutic target The human normal breast epithelial cells (MCF-10A; for BC [7]. basal subtype) and BC cell lines (MCF-7; Luminal A sub- MicroRNAs (miRNAs) could function as an impor- type and MDA-MB-231; basal subtype) were purchased tant class of posttranscriptional regulators through base from the American Type Culture Collection (Rockville, complementation with the 3′untranslated region (3′UTR) MD, USA). Dulbecco’s modified Eagle medium (Bio - of mRNAs, thereby altering the gene expression of car- chrom KG, Berlin, Germany) contained with 10% (v/v) cinogenic or tumor-inhibitory genes [8]. For example, fetal bovine serum (FBS; Sigma, San Francisco, CA, USA) miR-625-5p was reported to play significance roles in and 1% penicillin/streptomycin (Sigma) was used to malignant tumors. The upregulation of miR-625-5p incubate these cells in under standard culture conditions restricted cell growth of gastric cancer cells [9]. Addition-(5% CO , 37 °C). In addition, MCF-7 cells were cultured ally, the tumor-suppressive function also was reported in according to ATCC recommendation that 0.01  mg/mL BC; downregulation of miR-625 was found in BC tissues and cells, which was closely related to poor clinical out- comes of BC patients [10]. Insufficiently, the functions of miR-625-5p are not deeply investigated in BC. Table 1 Correlation of  clinicopathological features of  BC The transcriptional factor Zinc finger E-box binding patients with circMMP11expression levels homeobox-2 (ZEB2), belonging to ZEB family, is impli- Characteristics All cases circMMP11 p value cated in some signaling pathways involved in epithe- expression lial–mesenchymal transition (EMT) process [11]. Not High Low surprisingly, ZEB2 was reported to be essentially involved in EMT process of BC cells [12]. As previous report, the Age 0.7283 metastasis-associated function of ZEB2 has been dem- ≥ 45 20 9 11 onstrated in multiple tumors, including BC [13]. Analo- < 45 14 8 6 gously, Chen et al. [14] also found that miR-30a inhibited Tumor 0.7319 migration and invasion of nasopharyngeal carcinoma Left 18 10 8 cells by modulating ZEB2. Right 16 7 9 Therefore, this research was designed to inspect the Tumor size (cm) 0.1571 expression and regulatory mechanisms of circMMP11 ≥ 2 13 9 4 in BC. Based on above researches, we hypothesized that < 2 20 8 12 circMMP11 was involved in BC process via regulatory Molecular subtype 0.2962 networks of ceRNA. Luminal A 12 7 5 Luminal B 13 6 7 HER2E 2 2 0 Materials and methods TNBC 7 2 5 Patient specimens Family history of breast cancer 0.4905 We collected 34 paired BC specimens and the peracan- Yes 15 9 6 cer tissues from BC patients with surgical tumor resec- None 19 8 11 tions at Cancer Institute and Cancer Hospital, Chinese Lymph node metestasis 0.0039** Academy of Medical Sciences and Peking Union Medical Negative 21 6 15 College. All recruited BC patients completed the writ- Positive 13 11 2 ten informed consent. The removed tissues were stored TNM stage 0.0366* at − 80  °C after freeze in liquid nitrogen. The entire I–II 15 4 11 investigation was authorized by the Ethics Committee III–IV 19 13 6 of Cancer Institute and Cancer Hospital, Chinese Acad- emy of Medical Sciences and Peking Union Medical Col- HER2E Her2-enriched, TNBC Triple Negative Breast Cancer lege. The clinicopathologic features of BC patients were *P < 0.05, **P < 0.01, statistically significant Qi  et al. Cancer Cell Int (2021) 21:133 Page 3 of 12 human recombinant insulin was used to make the com- Hercules, CA, USA) by Clarity Western ECL Substrate plete growth medium. Kit (Bio-Rad). Real‑time quantitative polymerase chain reaction Transfection assay (RT‑qPCR) assay Small interfering RNA (siRNA) targeting circMMP11 Total RNA was extracted by RNA extraction kit (Thermo was amplified and synthesized by GenePharma (Shang - Fisher Scientific, Waltham, MA, USA) in the light of the hai, China). The sequences of si-circMMP11#1 were: user’s guideline. Following, total RNA was converted into 5′-AAG AAG AAG GUU UAU ACA CAC-3′; si-circ- complementary DNA (cDNA) by cDNA synthesis kit II MMP11#2, 5′-AGA AGA AGA AGG UUU AUA CAC-3′; (Exiqon, Woburn, MA, USA) or TransScript miRNA si-circMMP11#3; 5′-AAA ACA ACU GUG UUU AAU First-Strand cDNA Synthesis SuperMix (Transgen bio- GAC-3′; Negative siRNA: 5′-GGC CUA AAG UAG UAG tech, Beijing, China). The RT-qPCR assay was conducted CUA UTT-3′; ZEB2-overexpression vector (ZEB2) was under A Roche Light-Cycler (Roche, Basel, Switzerland) accomplished by amplifying cDNA of ZEB2 and subclon- with QuantiTect SYBR Green RT-PCR Kit (Qiagen, Dus- ing into the pcDNA vector (GenePharma). MiR-625-5p seldorf, NRW, Germany). In addition, endogenous small mimic (miR-625-5p), miR-NC, miR-625-5p inhibitor (in- nuclear RNA U6 was used as the internal reference for miR-625-5p), in-miR-con were purchased from Sangon miR-625-5p, while glyceraldehyde phosphate dehydro- (Shanghai, China). The oligonucleotides and vectors were genase (GAPDH) was used as the internal control for transferred into BC cells by Lipofectamine 2000 (Thermo −ΔΔCt circMMP11 and ZEB2 based on the 2 method. The Fisher Scientific). primers were shown as follows: 3‑(4,5‑Dimethylthiazol‑2‑yl)‑2,5‑diphenyl‑2H‑tetra ‑ circMMP11 (up 5′-CTT TTC GCA GCA CTG CTA zol‑3‑ium bromide (MTT) assay TCC-3′; down 5′-CCT TCC AGA GCC TTC ACC The cell viability of BC cells was determined by MTT TT-3′); assay. Briefly, the transfected BC cells were tiled to a miR-625-5p (up 5′-GCC GAG AGG GGG AAA GTT 96-well plate (3000 cells/well). Following, 20 µL of MTT CTA-3′; down, 5′-CAG TGC AGG GTC CGA GGT solution (Sigma) was added into cells and allowed to AT-3′); incubate for indicate times. After 4  h, the dimethyl sul- ZEB2 (up 5′-CAA GAG GCG CAA ACA AGC C-3′; foxide (Sigma) was used to dissolve formazan crystals. down 5′-GGT TGG CAA TAC CGT CAT CC-3′); The absorbance was measured under the multi-well scan - GAPDH (up 5′-GTG TTC CTA CCC CCA ATG TG-3′; ning spectrophotometer (Bio-Rad; wavelength: 450 nm). down 5′-CAT CGA AGG TGG AAG AGT GG-3′); Furthermore, the colony-forming assay was performed as U6 (up 5′-ATC CTT ACG CAC CCA GTC CA-3′; down previous description [15]. 5′-GAA CGC TTC ACG AAT TTG C-3′). Transwell assay Approximately 5 × 10 MCF-7 or MDA-MB-231 cells in serum-free medium were added into upper chamber Western blot assay of 24-well transwell chamber (8-µm pore size; BD Bio- Briefly, lysis buffer (Cell Signaling Technology, Danvers, sciences, San Jose, CA, USA), and the lower chamber MA, USA) was rapidly added to cells or tissues after was filled with complete medium containing 10% FBS. washing by pre-cooled phosphate buffer saline. After After 24 h, cells on the basal side of the membrane were quantifying and boiling, 40 µg of total protein was sub- stained by 0.1% crystal violet (Sigma) and photographed ject to 10% sodium dodecyl sulfate polyacrylamide gel under the microscope (Leica, Wetzlar, Germany; 100× electrophoresis and then transferred nitrocellulose magnification). For invasion assay, upper chamber was membranes (GE Healthcare, Piscataway, NJ, USA). After additionally covered with matrigel (BD Biosciences). blocking by 5% non-fat milk for 2  h, membranes were interacted with primary anti-bodies overnight at 4  °C, including anti-E-cadherin (#3195S; 1:1500 dilution), anti- Cell apoptosis assay Vimentin (#5741S; 1:1500 dilution), anti-ZEB2 (#3396S; Transfected BC cells were collected by trypsin, washed 1:1500 dilution), and anti-β-actin (#4970S; 1:1500 dilu- with pre-cooled phosphate buffer saline, and then re- tion; all purchased from Cell Signaling Technology). Fol- suspended in Binding Buffer containing Annexin V lowing, membranes were reacted to HRP-conjugated labeled with fluorescein isothiocyanate (FITC) and pro - secondary antibody (#7074S; 1:200 dilution; Cell Signal- pidium iodide (PI) (BestBio, Shanghai, China). Followed ing Technology). The relative expression of proteins was by incubated at 4 °C for 30 min, the apoptotic cells were detected under ChemiDoc MP imaging system (Bio-Rad, Qi et al. Cancer Cell Int (2021) 21:133 Page 4 of 12 determined on flow cytometric (Attune, Life Technolo - sense strand 5′-ccggAGA AGA AGA AGG TTT ATA gies, Darmstadt, Germany). C A C t t c aag ag aGT G TAT A A A C C T T C T T C T T C T T T T TTT GGT ACC-3′. antisense strand 5′-aattGGT ACC AAA AAA AGA AGA AGA AGG UUU AUA CAC TCT CTT GAA RNA pull‐down assay GTG TAT AAA CCT TCT TCT TCT-3′). In addition, ccgg The biotin-labeled RNA probe (Bio-circMMP11 probe) and ttaa was used to generate sticky-end by AgeI and was synthesized by GenePharma. MCF-7 or MDA- EcoRI, and ttcaagaga was used as stem-loop sequence. MB-231 cells were lysed by lysis buffer (Cell Signaling Technology). Subsequently, 100 µL of cell lysates was Statistical analysis incubated with Bio-circMMP11 probe at 4  °C for 4  h, All data were shown as mean ± standard deviation and with Bio-con as control, and the treated with 50 µL of performed in triplicate. Comparison of two treatment streptavidin beads (Cell Signaling Technology) at 4  °C groups was conducted by Student’s t-test, while sig- overnight to generate probe-bound dynabeads. The nificant difference of multiple groups was assessed by RNA complexes were extracted and measured by RT- one-way analysis of variance. Statistical analysis was con- qPCR assay. Similarly, the 3′end biotinylated miR-625-5p ducted by GraphPad Prism 7 (GraphPad, La Jolla, CA, (Riboio, Bio-miR-625-5p probe) was used to pull-down USA), and significant difference was considered if P-value circMMP11 and ZEB2 in cell lysates. less than 0.05. Pearson’s correlation analysis was used to reveal correlation relationship among circMMP11, miR- Dual‐luciferase report assay 625-5p, and ZEB2 in BC tissues. Based on bioinformatics prediction starbase (http://starb ase.sysu.edu.cn/), Circbank (http://www.circb ank.cn/), Results and Circular RNA interactome (https ://circi ntera ctome CircMMP11 and ZEB2 were overexpressed in BC tissues .irp.nia.nih.gov/), miR-625-5p was selected as a candidate and cells target of circMMP11. The complementary sequences To explore the role of circMMP11 in BC, the expres- between miR-625-5p and ZEB2 were presented by Tar- sion level of circMMP11 was quantified. The results of getscan (http://www.targe tscan .org/vert_72/). Partial RT-qPCR data suggested that circMMP11 were over- sequences of circMMP11 and 3′UTR of ZEB2 that con- expressed in BC tissues and cells when compared with tain the putative binding sites for miR-625-5p were paracancer tissues and MCF-10A cells (Fig.  1a, b). Fur- synthesized and cloned into luciferase report vectors thermore, ZEB2 also was significantly upregulated in BC (Ambion, Foster City, CA, USA). Subsequently, the BC tissues compared with adjacent normal tissues (Fig.  1c, cells were co-transfected with miR-625-5p mimic or d). The analogous results were confirmed in BC cells miR-NC and the wild or mutant type reports. The rela - that MCF-7 and MDA-MB-231 cells showed the relative tive firefly luciferase activity was determined under the higher expression of ZEB2 than MCF-10A cells (Fig.  1e, VICTOR2 fluorometry (PerkinElmer, Waltham, MA, f ). We hypothesized that circMMP11 and ZEB2 played USA) and normalized to Renilla luciferase activity. key roles in BC. Animal experiment Knockdown of circMMP11 constrained proliferation, migra‑ Six-week-old female BALB/c nude mice (Vital River tion, and invasion while induced apoptosis of BC cells Laboratory, Beijing, China) were housed under specific Subsequently, circMMP11 was inhibited by siRNAs tar- pathogen-free conditions. MCF-7 stably transfected with geting circMMP11 in MCF-7 and MDA-MB-231 cells. sh-circMMP11 (GeneCopoeia, Rockville, MD, USA) were 7 As shown in Fig.  2a, b, circMMP11 was downregulated inoculated into right back of nude mice (5 × 10 cells/ in si-circMMP11 groups compared with control group, mouse in 200 µL of complete growth medium; N = 6), especially in si-circMMP11#2 group. MTT indicated sup- with sh-NC as control. The xenograft volume was exam - pression of circMMP11 decreased cell viability of MCF-7 ined with digital calipers according to the volume = 1/2 2 and MDA-MB-231 cells (Fig.  2c, d). Likewise, colony- (length × width ). All of our animal experimental proto- forming ability of BC cells was inhibited by silencing of cols had been approved by the Institutional Animal Care circMMP11 (Fig.  2e). In addition, circMMP11 knock- and Use Committee of Cancer Institute and Cancer Hos- down obviously suppressed migration and invasion of pital, Chinese Academy of Medical Sciences and Peking BC cells by transwell assay (Fig.  2f, g). We also observed Union Medical College. The sh-circMMP11 sequence that suppression of circMMP11 increased cell apoptosis were amplified and then cloned into pLKO.1-Puro vector (Fig.  2h). The analysis results of western blot assay indi - (GeneCopoeia) between AgeI and EcoRI sites. Specific cated that inhibition of circMMP11 decreased Vimentin short hairpin RNA against circMMP11 (sh-circMMP11; while increased E-cadherin expression in MCF-7 and Qi  et al. Cancer Cell Int (2021) 21:133 Page 5 of 12 Fig. 1 The expression levels of circMMP11 and ZEB2 in breast cancer tissues and cells. a, b The relative expression level of circMMP11 was determined by RT-qPCR assay in breast cancer tissues and cells, along with in matched controls. c–f The RT-qPCR and western blot assays were performed to assess the expression of ZEB2 in breast cancer tissues and cells. *P < 0.05, **P < 0.01, ***P < 0.001 MDA-MB-231 cells (Fig.  2i, j). Collectively, circMMP11 MiR‑625‑5p, interacted with ZEB2, was a target of circ ‑ was aberrantly expressed as a tumor facilitator in BC. MMP11 To investigate target miRNAs of circMMP11, bioinfor- ZEB2 knockdown repressed proliferation, migration, matics software was used. As presented in Fig.  4a, the and invasion while induced apoptosis of BC cells overlap of candidate miRNAs contained miR-671-5p, Loss-of-function experiments were conducted in MCF-7 miR-625-5p, and miR-516p-5p. Interestingly, we found and MDA-MB-231 cells by siRNA targeting ZEB2. We that biotin-labeled circMMP11 could effectively pull- found that ZEB2 was downregulated in MCF-7 and down miR-625-5p in cell lysates from MCF-7 and MDA- MDA-MB-231 cells after transfection with si-ZEB2 MB-231 cells (Fig.  4b,  c). The binding regions between (Fig.  3a, b). Moreover, the inhibition of ZEB2 repressed miR-625-5p and circMMP11 or ZEB2 were presented the proliferation of MCF-7 and MDA-MB-231 cells in Fig.  4d, e. The luciferase activity was downregulated (Fig. 3c–e). The reduced numbers of migration and inva - in the group of co-transfection of miR-625-5p mimic sion were found in si-ZEB2 transfection group compared and circMMP11 WT in MCF-7 and MDA-MB-231 with control group (Fig.  3f, g). The data of flow cytom - cells, while circMMP11 MUT group showed no notable etry assay revealed that inhibition of ZEB2 enhanced cell change of luciferase activity (Fig.  4f, g). We also found apoptosis in BC cells (Fig. 3h). We also noticed that inhi- that circMMP11 was enriched by biotin-labeled miR- bition of ZEB2 increased the expression of E-cadherin 625-5p probe, suggesting that circMMP11 could inter- while decreased the expression of Vimentin in MCF-7 act with miR-625-5p (Fig.  4h). Similarly, overexpression and MDA-MB-231 cells (Fig. 3i, k). Therefore, these data of miR-625-5p decreased the luciferase activity of ZEB2 indicated that silencing of ZEB2 might impede the devel- 3′UTR WT; besides, ZEB2 was upregulated in Bio- opment of BC. miR-625-5p probe group compared with control group Qi et al. Cancer Cell Int (2021) 21:133 Page 6 of 12 Fig. 2 The effects of circMMP11 inhibition on proliferation, migration, invasion, and apoptosis of breast cancer cells. a, b The expression of circMMP11 was measured by RT-qPCR assay in MCF-7 and MDA-MB-231 cells transfected with si-NC, si-circMMP11#1, si-circMMP11#2, or si-circMMP11#3. c–j MCF-7 and MDA-MB-231 cells were transfected with si-NC or si-circMMP11#2. c–e The proliferation ability of MCF-7 and MDA-MB-231 cells were estimated by MTT and colony-forming assays. f, g Transwell assay was conducted to assess migration and invasion of MCF-7 and MDA-MB-231 cells. h The apoptotic cells were monitored by flow cytometry assay. i, j The protein expression levels of E-cadherin and Vimentin were quantified by western blot assay. **P < 0.01, ***P < 0.001 (Fig.  4i, k). What’s more, miR-625-5p was decreased in Western blot assay revealed that ZEB2 was obviously BC tissues and cells compared with matched controls upregulated in MCF-7 and MDA-MB-231 cells after (Fig. 4l, m). All data indicated that circMMP11 regulated transfected with ZEB2 (Fig.  5a, b). The silencing of circ - ZEB2 expression through sponging miR-625-5p. MMP11 inhibited the expression of ZEB2, which was rescued by transfection with ZEB2 (Fig.  5c, d). The sup - Overexpression of ZEB2 abolished circMMP11‑mediated pressive effect on cell proliferation in si-circMMP11#2- effects on BC cells tranfecting cells was abolished by overexpression of The regulatory relationship between circMMP11 and ZEB2 (Fig.  5e, g). The migration and invasion of MCF-7 ZEB2 was explored in MCF-7 and MDA-MB-231 cells. and MDA-MB-231 cells were suppressed by silencing of Qi  et al. Cancer Cell Int (2021) 21:133 Page 7 of 12 Fig. 3 Suppression of ZEB2 inhibited proliferation, migration, and invasion while induced apoptosis of breast cancer cells. a–k MCF-7 and MDA-MB-231 cells were transfected with si-NC or si-ZEB2. a, b The expression of ZEB2 was detected by RT-qPCR assay in MCF-7 and MDA-MB-231 cells. c–e MTT and colony-forming assays were performed in MCF-7 and MDA-MB-231 cells. f, g The migration and invasion of MCF-7 and MDA-MB-231 cells were assessed by transwell assay. h The apoptosis was examined by flow cytometry assay. i–k The western blot assay was used to test protein expression of E-cadherin and Vimentin in MCF-7 and MDA-MB-231 cells. **P < 0.01, ***P < 0.001 circMMP11, which was overturned by overexpression circMMP11 regulated ZEB2 expression in BC cells by of ZEB2 (Fig.  5h, i). The upregulation of ZEB2 abolished targeting miR-625-5p. circMMP11 inhibition-induced apoptosis in MCF-7 and MDA-MB-231 cells (Fig.  5j). The analysis results Inhibition of circMMP11 repressed tumorigenesis in vivo of western blot assay indicated that inhibition of circ- As shown in Fig.  7a, b, the knockdown of circMMP11 MMP11 decreased Vimentin while increased E-cadherin slowed the growth rate of tumor and decreased the expression, which was overturned by overexpression of weights of xenograft tumors when compared with con- ZEB2 (Fig.  5k, l). Conclusively, circMMP11 knockdown trol. The results of RT-qPCR assay indicated that circ - repressed proliferation, migration, and invasion while MMP11 and ZEB2 were decreased while miR-625-5p was induced apoptosis of BC cells through ZEB2. increased in sh-circMMP11 group compared with sh-NC group (Fig.  7c). In addition, the suppression of circ- CircMMP11 regulated miR‑625‑5p/ZEB2 axis in BC MMP11 also decreased the protein expression of ZEB2 in The correlation relationship among circMMP11, miR- dissected tumor tissues (Fig.  7d). This finding confirmed 625-5p, and ZEB2 was investigated in BC tissues. As pre- that silencing of circMMP11 repressed tumor growth sented in Fig. 6a, b, miR-625-5p was negatively correlated in vivo. with circMMP11 and ZEB2 expression in BC tissues. Furthermore, a positive correlation between circMMP11 Discussion and ZEB2 was revealed in BC tissues (Fig.  6c). Transfec- Currently, the regulatory networks of circRNA/micro- tion with in-miR-625-5p into MCF-7 and MDA-MB-231 RNA/mRNA was widely focused and reported in tumo- cells inhibited the expression of miR-625-5p (Fig.  6d). rigenesis [16]. Therefore, the molecular mechanisms The downregulation of ZEB2 in si-circMMP11#2-trans - of circMMP11 was investigated in BC. Consistent with fecing MCF-7 and MDA-MB-231 cells was abolished the previous conclusion [6, 7], our data indicated that by inhibition of miR-625-5p (Fig.  6e, f). In summary, Qi et al. Cancer Cell Int (2021) 21:133 Page 8 of 12 Fig. 4 CircMMP11 increased ZEB2 expression through sponging miR-625-5p. a Schematic illustration showed the overlap of the target miRNAs of circMMP11 predicted by Circbank, Starbase, and Circular RNA interactome. b, c The relative levels of miRNA candidates in MCF-7 and MDA-MB-231 cells lysates were examined by RT-qPCR after pulling down by circMMP11 probe. d Predicted binding regions between circMMP11 and miR-625-5p were shown. e The binding sites for miR-625-5p in 3′UTR of ZEB2 were presented. f–k The interaction relationships among circMMP11, miR-625-5p, and ZEB2 were confirmed by dual-luciferase report and RNA pull-down assays. l, m The expression level of miR-625-5p was measured by RT-qPCR assay in breast cancer tissues and cells, along with in matched controls. ***P < 0.001 circMMP11 was obviously higher in BC tissues than con- for BC [18]. MiR-625 played tumor-suppressive roles trol group. In addition, suppression of circMMP11 sup- in malignant tumor; miR-625-5p could target LRRC8E pressed cell growth and migration via directly targeting and highmobilitygroupAT-hook1 (HMGA1) in cervical miR-625-5p/ZEB2 axis. cancer and BC, respectively [19, 20]. Here, we demon- There are accumulating examples of miRNAs tar - strated that miR-625-5p targeted ZEB2 in BC cells. Of geting mRNA, thereby participating the initiation and course, other miRNAs could interact ZEB2 to regulate development of a large number of diseases, including the occurrence and progression of BC; multiple miR- BC [17]. Not surprisingly, a previous report implied NAs, including miR-30a [21], miR-653 [22], miR-448 that miR-625 was a possible and primary biomarker Qi  et al. Cancer Cell Int (2021) 21:133 Page 9 of 12 Fig. 5 Knockdown of circMMP11-mediated effects on breast cancer cells could be abolished by overexpression of ZEB2. a, b The overexpression effectiveness of ZEB2 was checked by RT-qPCR and western blot assays. c–l MCF-7 and MDA-MB-231 cells were transfected with si-NC, si-circMMP11#2, si-circMMP11#2 + pcDNA, or si-circMMP11#2 + ZEB2. c, d The expression levels of ZEB2 were assessed by RT-qPCR and western blot assays. e–g The proliferation was examined by MTT and colony-forming assays in MCF-7 and MDA-MB-231 cells. h, i The migration and invasion were determined by transwell assay. j The flow cytometry assay was performed in MCF-7 and MDA-MB-231 cells. i–k The protein expression levels of E-cadherin and Vimentin were measured by western blot assay in MCF-7 and MDA-MB-231 cells. *P < 0.05, **P < 0.01, ***P < 0.001 [23], and miR-124 [24] were identified to bind to the During the EMT process, decrease of E-cadherin and 3′UTR of ZEB2 in BC cells. increase of vimentin includes cellular mobility and inva- Furthermore, the high metastasis and recurrence of sion of tumor cells [27, 28]. Interestingly, ZEB2 exerts a BC impede clinical diagnosis, so inhibiting migration crucial effect on the metastasis of malignancy cells due to and invasion of BC cells is an important issue of current its ability in stimulation of EMT process [29, 30]. Previ- research [25]. EMT is one of the main mechanism, which ous reports have indicated that ZEB was associated with induces metastasis and dispersion of malignant tumors EMT in retinoblastoma, which promoted the invasion and drug-resistance while prevents apoptosis [26]. and metastasis of retinoblastoma cells [31]. Our results Qi et al. Cancer Cell Int (2021) 21:133 Page 10 of 12 Fig. 6 CircMMP11 regulated miR-625-5p/ZEB2 axis in breast cancer cells. a–c Pearson’s correlation analysis was used to reveal correlation relationship among circMMP11, miR-625-5p, and ZEB2 in breast cancer tissues. d The expression level of miR-625-5p was assessed by RT-qPCR in MCF-7 and MDA-MB-231 cells transfected with in-miR-con or in-miR-625-5p. e, f The expression levels of ZEB2 were measured by western blot assay in MCF-7 and MDA-MB-231 cells transfected with si-NC, si-circMMP11#2, si-circMMP11#2 + in-miR-co, or si-circMMP11#2 + in-miR-625-5p. **P < 0.01, ***P < 0.001 also indicated that ZEB2 partially attenuated the suppres- overexpressed in BC tissues and cells. CircMMP11 could sive influences of circMMP11 inhibition on cell growth directly bind to miR-625-5p, therefore promoting the and mobility of BC cells. In addition, ZEB2 was involved expression of ZEB2 via ceRNA mechanism, which pro- in EMT and downstream complex signaling pathways viding new insight into the pathogenesis and promising [32]. prognostic biomarker for BC. Nevertheless, certain limitations were faced during the study, including a limited sample size. In addition, cel- Conclusions lular heterogeneity play the key roles in cancer manage- In summary, circMMP11 was obviously upregulated ment, application of protein expression assay at single in BC tissues and cells. Mechanistic experiments sug- cell level was necessary to build objective conclusion in gested that circMMP11 exerted its tumor-promoting the future works. Although our results suggested that effects by modulating proliferation, migration, inva - the regulation of circMMP11/miR-625-5p/ZEB2 axis sion, and apoptosis of BC cells through targeting plays key roles in various biological functions of BC cells, miR-625-5p/ZEB2 axis, suggesting that circMMP11/ the involved downstream signal paths and regulatory miR-625-5p/ZEB2 axis could be used as diagnostic mechanisms have not been further investigated. Conclu- markers for BC. sively, our study provided evidence that circMMP11 was Qi  et al. Cancer Cell Int (2021) 21:133 Page 11 of 12 Fig. 7 Silencing of circMMP11 repressed tumor growth in vivo. a, b The growth curves and weights of xenograft tumors were shown. c The expression levels of circMMP11, miR-625-5p, and ZEB2 were estimated with RT-qPCR assay in dissected tumor tissues. d Western blot assay was used to assess the expression level of ZEB2. **P < 0.01, ***P < 0.001 Acknowledgements Informed consent None. Informed consent was obtained from all patients and proceed in accordance with ethical standards and legal standards. Authors’ contributions LQ contributed to the experimental work, fgures, and drafting of the manuscript. Competing interests BS made substantial contributions to the design of the study and data analysis. The authors declare that they have no competing interests. BY and SL assisted with the experiments and data analysis. LQ helped in drafting the manuscript as well as correction of the language. LQ and BS supervised all the Author details experiments. All authors have read and approved the fnal manuscript. Department of Breast Surgical Oncology, Cancer Institute and Cancer Hospi- tal, Chinese Academy of Medical Sciences and Peking Union Medical College, Funding No. 17 Panjiayuan Nanli, Chaoyang District, Beijing 100021, China. The 2nd This work was supported by Natural Science Foundation of China (No. Department of Breast Cancer, Tianjin Medical University Cancer Institute 81001187). and Hospital, Tianjin, China. Availability of data and materials Received: 4 November 2020 Accepted: 6 February 2021 Author declares that all data and material are available by corresponding author. Ethics approval and consent to participate The study design was approved by the Local Bioethical Committee. Qi et al. Cancer Cell Int (2021) 21:133 Page 12 of 12 References 18. Si H, Sun X, Chen Y, Cao Y, Chen S, Wang H, et al. Circulating microRNA- 1. Ghoncheh M, Pournamdar Z, Salehiniya H. Incidence and mortality and 92a and microRNA-21 as novel minimally invasive biomarkers for primary epidemiology of breast cancer in the world. Asian Pac J Cancer Prev. breast cancer. J Cancer Res Clin Oncol. 2013;139(2):223–9. 2016;17(S3):43–6. 19. Wang L, Zhong Y, Yang B, Zhu Y, Zhu X, Xia Z, et al. LINC00958 facilitates 2. 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CircMMP11 regulates proliferation, migration, invasion, and apoptosis of breast cancer cells through miR-625-5p/ZEB2 axis

Cancer Cell International , Volume 21 (1) – Feb 25, 2021

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Springer Journals
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Copyright © The Author(s) 2021
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1475-2867
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10.1186/s12935-021-01816-z
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Abstract

Background: Circular RNAs (circRNAs) have been demonstrated to play significant roles in regulating gene expres- sion in tumorigenesis, including breast cancer (BC). This study was designed to explore the role and underlying molecular mechanisms of circMMP11 in BC. Methods: The real-time quantitative polymerase chain reaction (RT-qPCR) assay was used for examining expression of circMMP11, microRNA-625-5p (miR-625-5p), and Zinc finger E-box binding homeobox-2 (ZEB2). The protein expres- sion of ZEB2, Vimentin, and E-cadherin was assessed by western blot assay. The proliferation ability of BC cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT ) and colony-forming assays. The transwell assay was used to measure migration and invasion of BC cells. The apoptotic cells were examined by flow cytometry assay. The interaction association among circMMP11, miR-625-5p, and ZEB2 was confirmed by RNA pull-down and dual-luciferase report assays. A xenograft experiment was established to clarify the role of circMMP11 silencing in vivo. Results: We found that circMMP11 and ZEB2 were overexpressed in BC tissues and cells compared with controls. The suppression of circMMP11 or ZEB2 repressed proliferation, migration, and invasion while induced apoptosis of BC cells. Additionally, miR-625-5p, interacted with ZEB2, was a target of circMMP11 in BC cells. CircMMP11 regulated the expression of ZEB2 by targeting miR-625-5p. Knockdown of circMMP11-mediated effects on BC cells could be abol- ished by overexpression of ZEB2. Consistently, silencing of circMMP11 impeded the tumor growth in vivo. Conclusions: CircMMP11/miR-625-5p/ZEB2 axis affected proliferation, migration, invasion, and apoptosis of BC cells through the mechanism of competing endogenous RNAs (ceRNA), indicating that circMMP11 was an oncogenic circRNA in BC. Keywords: circRNA, circMMP11, miR-625-5p, ZEB2, BC Introduction the statistic [2]. Although improved prognosis of patients Breast cancer (BC) is a common female cancer all over with BC had achieved, BC remains leading reason of the world [1]. The morbidity of BC has been rising, over cancer mortality among females [3]. Therefore, it is 2  million females were diagnosed with BC according to meaningful to look into pathogenesis and find diagnosis biomarkers of BC. Circular RNAs (circRNAs), a group of RNA molecules *Correspondence: uncsuj@163.com 1 with closed covalent loops, play critical roles in initia- Department of Breast Surgical Oncology, Cancer Institute and Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union tion and development of the multiple malignancies [4]. Medical College, No. 17 Panjiayuan Nanli, Chaoyang District, Accumulating evidence has revealed multiple charac- Beijing 100021, China terization of circRNAs, including microRNA sponges Full list of author information is available at the end of the article © The Author(s) 2021. 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://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Qi et al. Cancer Cell Int (2021) 21:133 Page 2 of 12 [5], CircMMP11 (hsa_circ_0062558) is derived from the presented in Table  1. BC patients were selected for fur- Matrix Metalloprotease 11 (MMP11) gene and located ther investigation based on two strict criteria: (1) patients on chr22 (24,125,597–24,126,503). A previous research were first diagnosed with BC; (2) patients provided the reported that a series of differentially expressed circR - written informed consent. Exclusion criteria: (1) patients NAs could function as novel biomarkers for BC, includ- received neoadjuvant therapy; (2) patients with other ing circMMP11 [6]. Interestingly, circMMP11 could complicated diseases. function as a competitive endogenous RNA (ceRNA) for miR-1204 to participate in the development of BC, indi- Cell lines and cell culture cating that circMMP11 was a potential therapeutic target The human normal breast epithelial cells (MCF-10A; for BC [7]. basal subtype) and BC cell lines (MCF-7; Luminal A sub- MicroRNAs (miRNAs) could function as an impor- type and MDA-MB-231; basal subtype) were purchased tant class of posttranscriptional regulators through base from the American Type Culture Collection (Rockville, complementation with the 3′untranslated region (3′UTR) MD, USA). Dulbecco’s modified Eagle medium (Bio - of mRNAs, thereby altering the gene expression of car- chrom KG, Berlin, Germany) contained with 10% (v/v) cinogenic or tumor-inhibitory genes [8]. For example, fetal bovine serum (FBS; Sigma, San Francisco, CA, USA) miR-625-5p was reported to play significance roles in and 1% penicillin/streptomycin (Sigma) was used to malignant tumors. The upregulation of miR-625-5p incubate these cells in under standard culture conditions restricted cell growth of gastric cancer cells [9]. Addition-(5% CO , 37 °C). In addition, MCF-7 cells were cultured ally, the tumor-suppressive function also was reported in according to ATCC recommendation that 0.01  mg/mL BC; downregulation of miR-625 was found in BC tissues and cells, which was closely related to poor clinical out- comes of BC patients [10]. Insufficiently, the functions of miR-625-5p are not deeply investigated in BC. Table 1 Correlation of  clinicopathological features of  BC The transcriptional factor Zinc finger E-box binding patients with circMMP11expression levels homeobox-2 (ZEB2), belonging to ZEB family, is impli- Characteristics All cases circMMP11 p value cated in some signaling pathways involved in epithe- expression lial–mesenchymal transition (EMT) process [11]. Not High Low surprisingly, ZEB2 was reported to be essentially involved in EMT process of BC cells [12]. As previous report, the Age 0.7283 metastasis-associated function of ZEB2 has been dem- ≥ 45 20 9 11 onstrated in multiple tumors, including BC [13]. Analo- < 45 14 8 6 gously, Chen et al. [14] also found that miR-30a inhibited Tumor 0.7319 migration and invasion of nasopharyngeal carcinoma Left 18 10 8 cells by modulating ZEB2. Right 16 7 9 Therefore, this research was designed to inspect the Tumor size (cm) 0.1571 expression and regulatory mechanisms of circMMP11 ≥ 2 13 9 4 in BC. Based on above researches, we hypothesized that < 2 20 8 12 circMMP11 was involved in BC process via regulatory Molecular subtype 0.2962 networks of ceRNA. Luminal A 12 7 5 Luminal B 13 6 7 HER2E 2 2 0 Materials and methods TNBC 7 2 5 Patient specimens Family history of breast cancer 0.4905 We collected 34 paired BC specimens and the peracan- Yes 15 9 6 cer tissues from BC patients with surgical tumor resec- None 19 8 11 tions at Cancer Institute and Cancer Hospital, Chinese Lymph node metestasis 0.0039** Academy of Medical Sciences and Peking Union Medical Negative 21 6 15 College. All recruited BC patients completed the writ- Positive 13 11 2 ten informed consent. The removed tissues were stored TNM stage 0.0366* at − 80  °C after freeze in liquid nitrogen. The entire I–II 15 4 11 investigation was authorized by the Ethics Committee III–IV 19 13 6 of Cancer Institute and Cancer Hospital, Chinese Acad- emy of Medical Sciences and Peking Union Medical Col- HER2E Her2-enriched, TNBC Triple Negative Breast Cancer lege. The clinicopathologic features of BC patients were *P < 0.05, **P < 0.01, statistically significant Qi  et al. Cancer Cell Int (2021) 21:133 Page 3 of 12 human recombinant insulin was used to make the com- Hercules, CA, USA) by Clarity Western ECL Substrate plete growth medium. Kit (Bio-Rad). Real‑time quantitative polymerase chain reaction Transfection assay (RT‑qPCR) assay Small interfering RNA (siRNA) targeting circMMP11 Total RNA was extracted by RNA extraction kit (Thermo was amplified and synthesized by GenePharma (Shang - Fisher Scientific, Waltham, MA, USA) in the light of the hai, China). The sequences of si-circMMP11#1 were: user’s guideline. Following, total RNA was converted into 5′-AAG AAG AAG GUU UAU ACA CAC-3′; si-circ- complementary DNA (cDNA) by cDNA synthesis kit II MMP11#2, 5′-AGA AGA AGA AGG UUU AUA CAC-3′; (Exiqon, Woburn, MA, USA) or TransScript miRNA si-circMMP11#3; 5′-AAA ACA ACU GUG UUU AAU First-Strand cDNA Synthesis SuperMix (Transgen bio- GAC-3′; Negative siRNA: 5′-GGC CUA AAG UAG UAG tech, Beijing, China). The RT-qPCR assay was conducted CUA UTT-3′; ZEB2-overexpression vector (ZEB2) was under A Roche Light-Cycler (Roche, Basel, Switzerland) accomplished by amplifying cDNA of ZEB2 and subclon- with QuantiTect SYBR Green RT-PCR Kit (Qiagen, Dus- ing into the pcDNA vector (GenePharma). MiR-625-5p seldorf, NRW, Germany). In addition, endogenous small mimic (miR-625-5p), miR-NC, miR-625-5p inhibitor (in- nuclear RNA U6 was used as the internal reference for miR-625-5p), in-miR-con were purchased from Sangon miR-625-5p, while glyceraldehyde phosphate dehydro- (Shanghai, China). The oligonucleotides and vectors were genase (GAPDH) was used as the internal control for transferred into BC cells by Lipofectamine 2000 (Thermo −ΔΔCt circMMP11 and ZEB2 based on the 2 method. The Fisher Scientific). primers were shown as follows: 3‑(4,5‑Dimethylthiazol‑2‑yl)‑2,5‑diphenyl‑2H‑tetra ‑ circMMP11 (up 5′-CTT TTC GCA GCA CTG CTA zol‑3‑ium bromide (MTT) assay TCC-3′; down 5′-CCT TCC AGA GCC TTC ACC The cell viability of BC cells was determined by MTT TT-3′); assay. Briefly, the transfected BC cells were tiled to a miR-625-5p (up 5′-GCC GAG AGG GGG AAA GTT 96-well plate (3000 cells/well). Following, 20 µL of MTT CTA-3′; down, 5′-CAG TGC AGG GTC CGA GGT solution (Sigma) was added into cells and allowed to AT-3′); incubate for indicate times. After 4  h, the dimethyl sul- ZEB2 (up 5′-CAA GAG GCG CAA ACA AGC C-3′; foxide (Sigma) was used to dissolve formazan crystals. down 5′-GGT TGG CAA TAC CGT CAT CC-3′); The absorbance was measured under the multi-well scan - GAPDH (up 5′-GTG TTC CTA CCC CCA ATG TG-3′; ning spectrophotometer (Bio-Rad; wavelength: 450 nm). down 5′-CAT CGA AGG TGG AAG AGT GG-3′); Furthermore, the colony-forming assay was performed as U6 (up 5′-ATC CTT ACG CAC CCA GTC CA-3′; down previous description [15]. 5′-GAA CGC TTC ACG AAT TTG C-3′). Transwell assay Approximately 5 × 10 MCF-7 or MDA-MB-231 cells in serum-free medium were added into upper chamber Western blot assay of 24-well transwell chamber (8-µm pore size; BD Bio- Briefly, lysis buffer (Cell Signaling Technology, Danvers, sciences, San Jose, CA, USA), and the lower chamber MA, USA) was rapidly added to cells or tissues after was filled with complete medium containing 10% FBS. washing by pre-cooled phosphate buffer saline. After After 24 h, cells on the basal side of the membrane were quantifying and boiling, 40 µg of total protein was sub- stained by 0.1% crystal violet (Sigma) and photographed ject to 10% sodium dodecyl sulfate polyacrylamide gel under the microscope (Leica, Wetzlar, Germany; 100× electrophoresis and then transferred nitrocellulose magnification). For invasion assay, upper chamber was membranes (GE Healthcare, Piscataway, NJ, USA). After additionally covered with matrigel (BD Biosciences). blocking by 5% non-fat milk for 2  h, membranes were interacted with primary anti-bodies overnight at 4  °C, including anti-E-cadherin (#3195S; 1:1500 dilution), anti- Cell apoptosis assay Vimentin (#5741S; 1:1500 dilution), anti-ZEB2 (#3396S; Transfected BC cells were collected by trypsin, washed 1:1500 dilution), and anti-β-actin (#4970S; 1:1500 dilu- with pre-cooled phosphate buffer saline, and then re- tion; all purchased from Cell Signaling Technology). Fol- suspended in Binding Buffer containing Annexin V lowing, membranes were reacted to HRP-conjugated labeled with fluorescein isothiocyanate (FITC) and pro - secondary antibody (#7074S; 1:200 dilution; Cell Signal- pidium iodide (PI) (BestBio, Shanghai, China). Followed ing Technology). The relative expression of proteins was by incubated at 4 °C for 30 min, the apoptotic cells were detected under ChemiDoc MP imaging system (Bio-Rad, Qi et al. Cancer Cell Int (2021) 21:133 Page 4 of 12 determined on flow cytometric (Attune, Life Technolo - sense strand 5′-ccggAGA AGA AGA AGG TTT ATA gies, Darmstadt, Germany). C A C t t c aag ag aGT G TAT A A A C C T T C T T C T T C T T T T TTT GGT ACC-3′. antisense strand 5′-aattGGT ACC AAA AAA AGA AGA AGA AGG UUU AUA CAC TCT CTT GAA RNA pull‐down assay GTG TAT AAA CCT TCT TCT TCT-3′). In addition, ccgg The biotin-labeled RNA probe (Bio-circMMP11 probe) and ttaa was used to generate sticky-end by AgeI and was synthesized by GenePharma. MCF-7 or MDA- EcoRI, and ttcaagaga was used as stem-loop sequence. MB-231 cells were lysed by lysis buffer (Cell Signaling Technology). Subsequently, 100 µL of cell lysates was Statistical analysis incubated with Bio-circMMP11 probe at 4  °C for 4  h, All data were shown as mean ± standard deviation and with Bio-con as control, and the treated with 50 µL of performed in triplicate. Comparison of two treatment streptavidin beads (Cell Signaling Technology) at 4  °C groups was conducted by Student’s t-test, while sig- overnight to generate probe-bound dynabeads. The nificant difference of multiple groups was assessed by RNA complexes were extracted and measured by RT- one-way analysis of variance. Statistical analysis was con- qPCR assay. Similarly, the 3′end biotinylated miR-625-5p ducted by GraphPad Prism 7 (GraphPad, La Jolla, CA, (Riboio, Bio-miR-625-5p probe) was used to pull-down USA), and significant difference was considered if P-value circMMP11 and ZEB2 in cell lysates. less than 0.05. Pearson’s correlation analysis was used to reveal correlation relationship among circMMP11, miR- Dual‐luciferase report assay 625-5p, and ZEB2 in BC tissues. Based on bioinformatics prediction starbase (http://starb ase.sysu.edu.cn/), Circbank (http://www.circb ank.cn/), Results and Circular RNA interactome (https ://circi ntera ctome CircMMP11 and ZEB2 were overexpressed in BC tissues .irp.nia.nih.gov/), miR-625-5p was selected as a candidate and cells target of circMMP11. The complementary sequences To explore the role of circMMP11 in BC, the expres- between miR-625-5p and ZEB2 were presented by Tar- sion level of circMMP11 was quantified. The results of getscan (http://www.targe tscan .org/vert_72/). Partial RT-qPCR data suggested that circMMP11 were over- sequences of circMMP11 and 3′UTR of ZEB2 that con- expressed in BC tissues and cells when compared with tain the putative binding sites for miR-625-5p were paracancer tissues and MCF-10A cells (Fig.  1a, b). Fur- synthesized and cloned into luciferase report vectors thermore, ZEB2 also was significantly upregulated in BC (Ambion, Foster City, CA, USA). Subsequently, the BC tissues compared with adjacent normal tissues (Fig.  1c, cells were co-transfected with miR-625-5p mimic or d). The analogous results were confirmed in BC cells miR-NC and the wild or mutant type reports. The rela - that MCF-7 and MDA-MB-231 cells showed the relative tive firefly luciferase activity was determined under the higher expression of ZEB2 than MCF-10A cells (Fig.  1e, VICTOR2 fluorometry (PerkinElmer, Waltham, MA, f ). We hypothesized that circMMP11 and ZEB2 played USA) and normalized to Renilla luciferase activity. key roles in BC. Animal experiment Knockdown of circMMP11 constrained proliferation, migra‑ Six-week-old female BALB/c nude mice (Vital River tion, and invasion while induced apoptosis of BC cells Laboratory, Beijing, China) were housed under specific Subsequently, circMMP11 was inhibited by siRNAs tar- pathogen-free conditions. MCF-7 stably transfected with geting circMMP11 in MCF-7 and MDA-MB-231 cells. sh-circMMP11 (GeneCopoeia, Rockville, MD, USA) were 7 As shown in Fig.  2a, b, circMMP11 was downregulated inoculated into right back of nude mice (5 × 10 cells/ in si-circMMP11 groups compared with control group, mouse in 200 µL of complete growth medium; N = 6), especially in si-circMMP11#2 group. MTT indicated sup- with sh-NC as control. The xenograft volume was exam - pression of circMMP11 decreased cell viability of MCF-7 ined with digital calipers according to the volume = 1/2 2 and MDA-MB-231 cells (Fig.  2c, d). Likewise, colony- (length × width ). All of our animal experimental proto- forming ability of BC cells was inhibited by silencing of cols had been approved by the Institutional Animal Care circMMP11 (Fig.  2e). In addition, circMMP11 knock- and Use Committee of Cancer Institute and Cancer Hos- down obviously suppressed migration and invasion of pital, Chinese Academy of Medical Sciences and Peking BC cells by transwell assay (Fig.  2f, g). We also observed Union Medical College. The sh-circMMP11 sequence that suppression of circMMP11 increased cell apoptosis were amplified and then cloned into pLKO.1-Puro vector (Fig.  2h). The analysis results of western blot assay indi - (GeneCopoeia) between AgeI and EcoRI sites. Specific cated that inhibition of circMMP11 decreased Vimentin short hairpin RNA against circMMP11 (sh-circMMP11; while increased E-cadherin expression in MCF-7 and Qi  et al. Cancer Cell Int (2021) 21:133 Page 5 of 12 Fig. 1 The expression levels of circMMP11 and ZEB2 in breast cancer tissues and cells. a, b The relative expression level of circMMP11 was determined by RT-qPCR assay in breast cancer tissues and cells, along with in matched controls. c–f The RT-qPCR and western blot assays were performed to assess the expression of ZEB2 in breast cancer tissues and cells. *P < 0.05, **P < 0.01, ***P < 0.001 MDA-MB-231 cells (Fig.  2i, j). Collectively, circMMP11 MiR‑625‑5p, interacted with ZEB2, was a target of circ ‑ was aberrantly expressed as a tumor facilitator in BC. MMP11 To investigate target miRNAs of circMMP11, bioinfor- ZEB2 knockdown repressed proliferation, migration, matics software was used. As presented in Fig.  4a, the and invasion while induced apoptosis of BC cells overlap of candidate miRNAs contained miR-671-5p, Loss-of-function experiments were conducted in MCF-7 miR-625-5p, and miR-516p-5p. Interestingly, we found and MDA-MB-231 cells by siRNA targeting ZEB2. We that biotin-labeled circMMP11 could effectively pull- found that ZEB2 was downregulated in MCF-7 and down miR-625-5p in cell lysates from MCF-7 and MDA- MDA-MB-231 cells after transfection with si-ZEB2 MB-231 cells (Fig.  4b,  c). The binding regions between (Fig.  3a, b). Moreover, the inhibition of ZEB2 repressed miR-625-5p and circMMP11 or ZEB2 were presented the proliferation of MCF-7 and MDA-MB-231 cells in Fig.  4d, e. The luciferase activity was downregulated (Fig. 3c–e). The reduced numbers of migration and inva - in the group of co-transfection of miR-625-5p mimic sion were found in si-ZEB2 transfection group compared and circMMP11 WT in MCF-7 and MDA-MB-231 with control group (Fig.  3f, g). The data of flow cytom - cells, while circMMP11 MUT group showed no notable etry assay revealed that inhibition of ZEB2 enhanced cell change of luciferase activity (Fig.  4f, g). We also found apoptosis in BC cells (Fig. 3h). We also noticed that inhi- that circMMP11 was enriched by biotin-labeled miR- bition of ZEB2 increased the expression of E-cadherin 625-5p probe, suggesting that circMMP11 could inter- while decreased the expression of Vimentin in MCF-7 act with miR-625-5p (Fig.  4h). Similarly, overexpression and MDA-MB-231 cells (Fig. 3i, k). Therefore, these data of miR-625-5p decreased the luciferase activity of ZEB2 indicated that silencing of ZEB2 might impede the devel- 3′UTR WT; besides, ZEB2 was upregulated in Bio- opment of BC. miR-625-5p probe group compared with control group Qi et al. Cancer Cell Int (2021) 21:133 Page 6 of 12 Fig. 2 The effects of circMMP11 inhibition on proliferation, migration, invasion, and apoptosis of breast cancer cells. a, b The expression of circMMP11 was measured by RT-qPCR assay in MCF-7 and MDA-MB-231 cells transfected with si-NC, si-circMMP11#1, si-circMMP11#2, or si-circMMP11#3. c–j MCF-7 and MDA-MB-231 cells were transfected with si-NC or si-circMMP11#2. c–e The proliferation ability of MCF-7 and MDA-MB-231 cells were estimated by MTT and colony-forming assays. f, g Transwell assay was conducted to assess migration and invasion of MCF-7 and MDA-MB-231 cells. h The apoptotic cells were monitored by flow cytometry assay. i, j The protein expression levels of E-cadherin and Vimentin were quantified by western blot assay. **P < 0.01, ***P < 0.001 (Fig.  4i, k). What’s more, miR-625-5p was decreased in Western blot assay revealed that ZEB2 was obviously BC tissues and cells compared with matched controls upregulated in MCF-7 and MDA-MB-231 cells after (Fig. 4l, m). All data indicated that circMMP11 regulated transfected with ZEB2 (Fig.  5a, b). The silencing of circ - ZEB2 expression through sponging miR-625-5p. MMP11 inhibited the expression of ZEB2, which was rescued by transfection with ZEB2 (Fig.  5c, d). The sup - Overexpression of ZEB2 abolished circMMP11‑mediated pressive effect on cell proliferation in si-circMMP11#2- effects on BC cells tranfecting cells was abolished by overexpression of The regulatory relationship between circMMP11 and ZEB2 (Fig.  5e, g). The migration and invasion of MCF-7 ZEB2 was explored in MCF-7 and MDA-MB-231 cells. and MDA-MB-231 cells were suppressed by silencing of Qi  et al. Cancer Cell Int (2021) 21:133 Page 7 of 12 Fig. 3 Suppression of ZEB2 inhibited proliferation, migration, and invasion while induced apoptosis of breast cancer cells. a–k MCF-7 and MDA-MB-231 cells were transfected with si-NC or si-ZEB2. a, b The expression of ZEB2 was detected by RT-qPCR assay in MCF-7 and MDA-MB-231 cells. c–e MTT and colony-forming assays were performed in MCF-7 and MDA-MB-231 cells. f, g The migration and invasion of MCF-7 and MDA-MB-231 cells were assessed by transwell assay. h The apoptosis was examined by flow cytometry assay. i–k The western blot assay was used to test protein expression of E-cadherin and Vimentin in MCF-7 and MDA-MB-231 cells. **P < 0.01, ***P < 0.001 circMMP11, which was overturned by overexpression circMMP11 regulated ZEB2 expression in BC cells by of ZEB2 (Fig.  5h, i). The upregulation of ZEB2 abolished targeting miR-625-5p. circMMP11 inhibition-induced apoptosis in MCF-7 and MDA-MB-231 cells (Fig.  5j). The analysis results Inhibition of circMMP11 repressed tumorigenesis in vivo of western blot assay indicated that inhibition of circ- As shown in Fig.  7a, b, the knockdown of circMMP11 MMP11 decreased Vimentin while increased E-cadherin slowed the growth rate of tumor and decreased the expression, which was overturned by overexpression of weights of xenograft tumors when compared with con- ZEB2 (Fig.  5k, l). Conclusively, circMMP11 knockdown trol. The results of RT-qPCR assay indicated that circ - repressed proliferation, migration, and invasion while MMP11 and ZEB2 were decreased while miR-625-5p was induced apoptosis of BC cells through ZEB2. increased in sh-circMMP11 group compared with sh-NC group (Fig.  7c). In addition, the suppression of circ- CircMMP11 regulated miR‑625‑5p/ZEB2 axis in BC MMP11 also decreased the protein expression of ZEB2 in The correlation relationship among circMMP11, miR- dissected tumor tissues (Fig.  7d). This finding confirmed 625-5p, and ZEB2 was investigated in BC tissues. As pre- that silencing of circMMP11 repressed tumor growth sented in Fig. 6a, b, miR-625-5p was negatively correlated in vivo. with circMMP11 and ZEB2 expression in BC tissues. Furthermore, a positive correlation between circMMP11 Discussion and ZEB2 was revealed in BC tissues (Fig.  6c). Transfec- Currently, the regulatory networks of circRNA/micro- tion with in-miR-625-5p into MCF-7 and MDA-MB-231 RNA/mRNA was widely focused and reported in tumo- cells inhibited the expression of miR-625-5p (Fig.  6d). rigenesis [16]. Therefore, the molecular mechanisms The downregulation of ZEB2 in si-circMMP11#2-trans - of circMMP11 was investigated in BC. Consistent with fecing MCF-7 and MDA-MB-231 cells was abolished the previous conclusion [6, 7], our data indicated that by inhibition of miR-625-5p (Fig.  6e, f). In summary, Qi et al. Cancer Cell Int (2021) 21:133 Page 8 of 12 Fig. 4 CircMMP11 increased ZEB2 expression through sponging miR-625-5p. a Schematic illustration showed the overlap of the target miRNAs of circMMP11 predicted by Circbank, Starbase, and Circular RNA interactome. b, c The relative levels of miRNA candidates in MCF-7 and MDA-MB-231 cells lysates were examined by RT-qPCR after pulling down by circMMP11 probe. d Predicted binding regions between circMMP11 and miR-625-5p were shown. e The binding sites for miR-625-5p in 3′UTR of ZEB2 were presented. f–k The interaction relationships among circMMP11, miR-625-5p, and ZEB2 were confirmed by dual-luciferase report and RNA pull-down assays. l, m The expression level of miR-625-5p was measured by RT-qPCR assay in breast cancer tissues and cells, along with in matched controls. ***P < 0.001 circMMP11 was obviously higher in BC tissues than con- for BC [18]. MiR-625 played tumor-suppressive roles trol group. In addition, suppression of circMMP11 sup- in malignant tumor; miR-625-5p could target LRRC8E pressed cell growth and migration via directly targeting and highmobilitygroupAT-hook1 (HMGA1) in cervical miR-625-5p/ZEB2 axis. cancer and BC, respectively [19, 20]. Here, we demon- There are accumulating examples of miRNAs tar - strated that miR-625-5p targeted ZEB2 in BC cells. Of geting mRNA, thereby participating the initiation and course, other miRNAs could interact ZEB2 to regulate development of a large number of diseases, including the occurrence and progression of BC; multiple miR- BC [17]. Not surprisingly, a previous report implied NAs, including miR-30a [21], miR-653 [22], miR-448 that miR-625 was a possible and primary biomarker Qi  et al. Cancer Cell Int (2021) 21:133 Page 9 of 12 Fig. 5 Knockdown of circMMP11-mediated effects on breast cancer cells could be abolished by overexpression of ZEB2. a, b The overexpression effectiveness of ZEB2 was checked by RT-qPCR and western blot assays. c–l MCF-7 and MDA-MB-231 cells were transfected with si-NC, si-circMMP11#2, si-circMMP11#2 + pcDNA, or si-circMMP11#2 + ZEB2. c, d The expression levels of ZEB2 were assessed by RT-qPCR and western blot assays. e–g The proliferation was examined by MTT and colony-forming assays in MCF-7 and MDA-MB-231 cells. h, i The migration and invasion were determined by transwell assay. j The flow cytometry assay was performed in MCF-7 and MDA-MB-231 cells. i–k The protein expression levels of E-cadherin and Vimentin were measured by western blot assay in MCF-7 and MDA-MB-231 cells. *P < 0.05, **P < 0.01, ***P < 0.001 [23], and miR-124 [24] were identified to bind to the During the EMT process, decrease of E-cadherin and 3′UTR of ZEB2 in BC cells. increase of vimentin includes cellular mobility and inva- Furthermore, the high metastasis and recurrence of sion of tumor cells [27, 28]. Interestingly, ZEB2 exerts a BC impede clinical diagnosis, so inhibiting migration crucial effect on the metastasis of malignancy cells due to and invasion of BC cells is an important issue of current its ability in stimulation of EMT process [29, 30]. Previ- research [25]. EMT is one of the main mechanism, which ous reports have indicated that ZEB was associated with induces metastasis and dispersion of malignant tumors EMT in retinoblastoma, which promoted the invasion and drug-resistance while prevents apoptosis [26]. and metastasis of retinoblastoma cells [31]. Our results Qi et al. Cancer Cell Int (2021) 21:133 Page 10 of 12 Fig. 6 CircMMP11 regulated miR-625-5p/ZEB2 axis in breast cancer cells. a–c Pearson’s correlation analysis was used to reveal correlation relationship among circMMP11, miR-625-5p, and ZEB2 in breast cancer tissues. d The expression level of miR-625-5p was assessed by RT-qPCR in MCF-7 and MDA-MB-231 cells transfected with in-miR-con or in-miR-625-5p. e, f The expression levels of ZEB2 were measured by western blot assay in MCF-7 and MDA-MB-231 cells transfected with si-NC, si-circMMP11#2, si-circMMP11#2 + in-miR-co, or si-circMMP11#2 + in-miR-625-5p. **P < 0.01, ***P < 0.001 also indicated that ZEB2 partially attenuated the suppres- overexpressed in BC tissues and cells. CircMMP11 could sive influences of circMMP11 inhibition on cell growth directly bind to miR-625-5p, therefore promoting the and mobility of BC cells. In addition, ZEB2 was involved expression of ZEB2 via ceRNA mechanism, which pro- in EMT and downstream complex signaling pathways viding new insight into the pathogenesis and promising [32]. prognostic biomarker for BC. Nevertheless, certain limitations were faced during the study, including a limited sample size. In addition, cel- Conclusions lular heterogeneity play the key roles in cancer manage- In summary, circMMP11 was obviously upregulated ment, application of protein expression assay at single in BC tissues and cells. Mechanistic experiments sug- cell level was necessary to build objective conclusion in gested that circMMP11 exerted its tumor-promoting the future works. Although our results suggested that effects by modulating proliferation, migration, inva - the regulation of circMMP11/miR-625-5p/ZEB2 axis sion, and apoptosis of BC cells through targeting plays key roles in various biological functions of BC cells, miR-625-5p/ZEB2 axis, suggesting that circMMP11/ the involved downstream signal paths and regulatory miR-625-5p/ZEB2 axis could be used as diagnostic mechanisms have not been further investigated. Conclu- markers for BC. sively, our study provided evidence that circMMP11 was Qi  et al. Cancer Cell Int (2021) 21:133 Page 11 of 12 Fig. 7 Silencing of circMMP11 repressed tumor growth in vivo. a, b The growth curves and weights of xenograft tumors were shown. c The expression levels of circMMP11, miR-625-5p, and ZEB2 were estimated with RT-qPCR assay in dissected tumor tissues. d Western blot assay was used to assess the expression level of ZEB2. **P < 0.01, ***P < 0.001 Acknowledgements Informed consent None. Informed consent was obtained from all patients and proceed in accordance with ethical standards and legal standards. Authors’ contributions LQ contributed to the experimental work, fgures, and drafting of the manuscript. Competing interests BS made substantial contributions to the design of the study and data analysis. The authors declare that they have no competing interests. BY and SL assisted with the experiments and data analysis. LQ helped in drafting the manuscript as well as correction of the language. LQ and BS supervised all the Author details experiments. All authors have read and approved the fnal manuscript. Department of Breast Surgical Oncology, Cancer Institute and Cancer Hospi- tal, Chinese Academy of Medical Sciences and Peking Union Medical College, Funding No. 17 Panjiayuan Nanli, Chaoyang District, Beijing 100021, China. The 2nd This work was supported by Natural Science Foundation of China (No. Department of Breast Cancer, Tianjin Medical University Cancer Institute 81001187). and Hospital, Tianjin, China. Availability of data and materials Received: 4 November 2020 Accepted: 6 February 2021 Author declares that all data and material are available by corresponding author. Ethics approval and consent to participate The study design was approved by the Local Bioethical Committee. Qi et al. Cancer Cell Int (2021) 21:133 Page 12 of 12 References 18. Si H, Sun X, Chen Y, Cao Y, Chen S, Wang H, et al. Circulating microRNA- 1. Ghoncheh M, Pournamdar Z, Salehiniya H. Incidence and mortality and 92a and microRNA-21 as novel minimally invasive biomarkers for primary epidemiology of breast cancer in the world. Asian Pac J Cancer Prev. breast cancer. J Cancer Res Clin Oncol. 2013;139(2):223–9. 2016;17(S3):43–6. 19. Wang L, Zhong Y, Yang B, Zhu Y, Zhu X, Xia Z, et al. LINC00958 facilitates 2. 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