MiR-591 functions as tumor suppressor in breast cancer by targeting TCF4 and inhibits Hippo-YAP/TAZ signaling pathway

A total of 78 pairs of human breast cancer tissue samples and corresponding adjacent normal tissues were obtained from patients who underwent resection at Department of Breast Surgery, The First Affiliated Hospital of Jinan University between August 2014 and July 2016. All of the patients were female and the median age of the patients was 50 years (range from 28 to 76 years old). The study was approved by the Ethical Committee of The First Affiliated Hospital of Jinan University (the protocol number IACUC-20170602-01) and all of the patients provided written informed consent. Pathological classification and staging were based on the 2007 International Breast Cancer Typing Guidelines.

Four human BC cell lines MCF-7, MDA-MB-231, T-47D, SKBR3 and a human normal breast epithelial cell line MCF-10A were purchased from the Institute of Biochemistry and Cell Biology, Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified 5% CO₂ incubator at 37 °C.

Chemically synthesized miR-591 mimic, miR-591 inhibitor or miR-negative control (NC), pcDNA-vector and pcDNA-TCF4 were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Cells were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer’s protocol.

Total RNA was extracted from tissues and cell lines using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). The total RNA was reverse transcribed to cDNA using the Prime Script RT reagent kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer’s protocol. The qRT-PCR reaction was performed using the SYBR Premix Ex Taq (Takara Bio, Inc., Otsu, Japan) on an ABI 7500 Sequence Detection system (Applied Biosystems). The U6 snRNA was used as an internal control. The thermocycling conditions was as follow: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s, and one cycle of 95 °C for 15 s, 60 °C for 60 s and 95 °C for 15 s for dissociation. The relative mRNA expression was calculated using the 2^−∆∆Cq methods.

Cell proliferation ability were assessed by CCK8 cell proliferation. Transfected cells (3 × 10³ cells/per well) were seeded in six-well plates. Cells were cultured for 0, 24, 48 and 72 h. Then, CCK8 solution (10 µl) was added into each well to incubate for 2 h at 37 °C, the plates were detected at indicated time (0, 24, 48 and 72 h) using a microplate reader at a wavelength of 450 nm.

After cells were transfected with miR-591 mimic, miR-591 inhibitor or miR-NC for 24 h, cells were seeded in Matrigel-coated chambers (8 μm pore size, BD Bioscience, USA). 1 × 10⁵ MCF-7 cells or MDA-MB-231 cells were added in the upper chamber with serum-free medium. Medium containing 10% FBS was placed in the lower chambers to act as a chemoattractant. After 48 h, cells were fixed and then stained with 0.1% crystal violet. Cell invasing number was assessed at five fields under a light microscope (Ti; Nikon Corporation, Tokyo, Japan) at magnification of 200×.

Potential miR-591 binding sites were predicted using TargetScan (http://​www.​targetscan.​org). The sequence of TCF4 with the wild-type or mutant 3′UTR seed region was synthesized and cloned into a pMIR-REPORT luciferase vector (Applied Biosystems; Thermo Fisher Scientific, Inc.). Then, The TCF4 with the wild-type or mutant 3′UTR pMIR-REPORT vector was co-transfected into MCF-7 cells with miR-591 mimic or miR-NC using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Firefly luciferase activity was detected at 48 h after transfection normalized against Renilla luciferase activity by using the Dual Luciferase Reporter Assay system (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocol.

Total protein was extracted from transfected cells by using a lysis buffer containing 150 mmol/l NaCl, 50 mmol/l Tris (pH 7.4), 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Protein concentration was detected by using bicinchoninic acid assay kit (BCA) (Beyotime Institute of Biotechnology, Haimen, China). A total 30 µg protein was added on 10% polyacrylamide gel and transfer to a polyvinylidene (PVDF) membrane. The membranes were blocked with 5% skimmed milk (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 1 h. Then, the membranes were incubated with primary antibody against TCF4 (Abcam, Cambridge, UK), YAP1 (Santa Cruz Biotechnology, Inc.), LATS1 (Santa Cruz Biotechnology, Inc.) and GAPDH (Cell Signaling Technology Inc.) at 4 °C overnight. Subsequently, the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Epitomics; Abcam, Cambridge, UK) at room temperature. The membranes were detected by using an enhanced chemiluminescence detection kit (Pierce; Thermo Fisher Scientific, Inc.) and the GAPDH expression was used as an internal control. Each experiment was repeated three times.

All animal experiments were conducted in accordance with current Chinese regulations and standards regarding the use of laboratory animals, and all animal procedures were approved by The First Affiliated Hospital of Jinan University Animal Care and Use Committee (the protocol number IACUC-20180101-01). 4-week-old BABL/c nude mice were purchased from the Center of Laboratory Animal Science of Guangdong (Guangzhou, China). Xenograft tumors were generated by subcutaneous injection of 1 × 10⁶ stable MCF-7 cells of miR-vector and miR-mimic on the hindlimbs. After 4 weeks, all mice were euthanized by dislocating the cervical spine. Tumor size was measured by a slide caliper (volume = length × width × height).

To explore the clinical role in breast cancer, we examined the miR-591 expression in breast cancer tissues and adjacent normal tissues by qRT-PCR methods. The results observed that miR-591 expression is dramatically downregulated in breast cancer than that in adjacent normal tissues (Fig. 1a). The mean level of miR-591 expression in breast cancer tissues was used as a threshold to divide patients into two groups (lower and higher expression groups). Association between miR-591 expression and clinical data of patients with breast cancer was analyzed. Results showed that miR-591 expression significantly correlated with advanced TNM stage (P = 0.011) and lymph node metastasis (P = 0.005) of patients (Table 1).

Furthermore, we analyzed the expression of miR-591 in four human BC cell lines MCF-7, MDA-MB-231, T-47D, SKBR3 and a human normal breast epithelial cell line MCF-10A. The qRT-PCR assay results indicated that miR-591 expression was lower in breast cancer cells compared to MCF-10A cells (Fig. 1b). To evaluate the effects of miR-591 expression on cell proliferation and invasion, we performed gain and loss function assays. The results showed that miR-591 mimic transfected MCF-7 and MDA-MB-231 cells had a dramatic higher miR-591 expression, but miR-591 inhibitor transfected MCF-7 and MDA-MB-231 cells presented a lower miR-591 expression, compared to corresponding controls, respectively (Fig. 1c, d). Subsequently, the cell proliferation and invasion ability of MCF-7 and SKBR3 cells were determined using CCK8 assay and transwell assays. MCF-7 and SKBR3 cells transfected with miR-591 mimic were significantly reduced cell proliferation ability compared with the control cells at 48 h and 72 h, whereas cells transfected with the miR-591 inhibitor were significantly increased cell proliferation ability compared with the control cells (Fig. 2a, b). Furthermore, transfection of miR-591 mimic in MCF-7 and SKBR3 cells significantly inhibited cell invasion ability compared with the control cells at 48 h, whereas transfection of miR-591 inhibitor in MCF-7 and SKBR3 cells significantly enhancing cell invasion ability compared with the control cells (Fig. 2c, d). Thus, these results indicated that miR-591 inhibited cell proliferation and invasion ability of breast cancer.

Furthermore, xenograft tumors were generated by subcutaneous injection of 1 × 10⁶ stable MCF-7 cells of miR-vector and miR-mimic on the hindlimbs. After 4 weeks, all mice were euthanized by dislocating the cervical spine. Tumor size was measured by a slide caliper (volume = length × width × height). Our results demonstrated that miR-591 mimic group showed much smaller tumors volume than that in miR-NC group (Fig. 3a). In addition, the tumor volume grown slower in miR-591 mimic group compared to that in miR-NC group (Fig. 3b). Thus, these results indicated that upregulation of miR-591 suppressed the proliferation of breast cancer cells in vivo.

By online databases (TargetScan and miRDB) predicted relative target of miR-591, we found that Transcription factor 4 (TCF4) was identified as a potential target of miR-591 (Fig. 4a). Furthermore, we co-transfected of TCF4-WT and miR-591 mimic in MCF-7 cells resulted in lower luciferase activity, while co-transfected of TCF4-MUT and miR-591 mimic showed no change in luciferase activity (Fig. 4b). Moreover, we detected the mRNA expression of TCF4 after upregulation of miR-591 or downregulation of miR-591 in MCF-7 or SKBR3 cells by qRT-PCR analysis. We found that transfection of miR-591 mimic in MCF-7 or SKBR3 cells significantly downregulated the mRNA expression of TCF4 (Fig. 4c, d). Transfection of MiR-591 inhibitor in MCF-7 cells resulted in upregulation of TCF4 expression (Fig. 4c, d). In additional, we also demonstrated that transfection of miR-591 mimic significantly downregulated the protein expression of TCF4 in MCF-7 or SKBR3 cells compared to the control groups (Fig. 4e, f). Thus, these results indicated that TCF4 was a direct target of miR-591 in breast cells.

To determine whether miR-591 suppresses cancer cell proliferation and invasion by regulating TCF4. We constructed TCF4 over-expressing vector and transfected into MCF-7 cells. Over-expression of TCF4 was confirmed by qRT-PCR analysis (Fig. 5a). The CCK8 cell proliferation and transwell assays showed that miR-591-mediated suppression of cell proliferation and invasion was partially rescued by overexpressed TCF4 in MCF-7 cells (Fig. 5b, d). Together, these data indicate that miR-591 suppresses cell proliferation and invasion by down-regulating TCF4.

Hippo-YAP signaling pathway has been reported to involved in tumor progression in breast cancer [13]. We detected the YAP expression by upregulation of miR-591 in MCF-7 and SKBR3 cells. The western blot analysis showed that YAP1 expression was downregulated after miR-591 upregulation in MCF-7 or SKBR3 cells compared to the control groups (Fig. 6a, b). However, LAST1 expression was upregulated after miR-591 upregulation in MCF-7 or SKBR3 cells compared to the control groups (Fig. 6a, b). Thus, these results indicated that miR-591 inhibited Hippo-YAP signaling pathway in breast cancer.