miR-148a-3p suppresses epithelial ovarian cancer progression primarily by targeting c-Met.

MicroRNAs (miRNAs) are a group of small non-coding RNAs that modulate post-transcriptional gene expression. It has been demonstrated that various miRNAs may be expressed at different levels in different types of tumors. The present study assessed the role of microRNA-148a-3p (miR-148a-3p) in epithelial ovarian cancer (EOC). The results demonstrated that miR-148a-3p was decreased in EOC tissues and that a lower miRa-148-3p concentration was associated with a higher overall survival rate. Transfection of miR-148a-3p suppressed the invasive and proliferative capacity of SKOV3 cells. The induced overexpression of miR-148a-3p significantly inhibited the relative luciferase activity of the pmirGLO-c-Met-3'untranslated region compared with an empty vector. In addition, c-Met silencing led to a decrease in the invasive and proliferative capacity of EOC cells. The inhibition of miR-148a-3p did not increase the invasiveness of SKOV3 cells, even when c-Met was silenced. To the best of our knowledge, the present study is the first to demonstrate that miR-148a-3p expression is decreased in EOC cancer tissues and cell lines. The present study therefore demonstrated that miR-148a-3p may serve as a tumor suppressor in EOC by targeting c-Met.

Introduction

Ovarian cancer, including epithelial ovarian cancer (EOC), has a high prevalence and is one of the most severe types of cancer affecting the female reproductive tract (1,2). Among ovarian malignancies, EOC is a primary cause of gynecological cancer-associated mortality, due to the increased metastatic nature of EOC cells (3). Previous studies have demonstrated that ovarian tumor cells migrate from primary sites to the peritoneal cavity (4,5). The diagnosis and treatment of EOC remains challenging due to a lack of reliable diagnostic biomarkers (6,7). Therefore, it is important to elucidate the underlying mechanisms that drive metastasis and chemoresistance in EOC.

During the progression of various tumors, abnormal cancer cell metastasis is attributed to a high rate of relapse. The hepatocyte growth factor receptor tyrosine-protein kinase Met (c-Met) enhances cancer cell motility and invasion (8). Previous studies have demonstrated that the c-Met proto-oncogene (MET), also known as the hepatocyte growth factor receptor, is upregulated in various tumors (2,9). Furthermore, patients with metastatic EOC exhibit increased levels of MET; ~10% of Caucasian patients with EOC possess multiple copies of MET and it is associated with unfavorable clinical outcomes (8,10).

It has been demonstrated that microRNAs (miRNAs) serve a primary role in the regulation of MET expression. miRNAs are small non-coding RNAs, ~22 nucleotides in length, that modulate post-transcriptional gene expression (8,11). miRNAs are aberrantly expressed in ovarian cancer. It has been demonstrated that miRNA-30a-5p overexpression increases the proliferation, colony formation, migration and invasion of ovarian cancer cells (12). miRNA-137 and miRNA-34a suppress the invasiveness and sphere-forming ability of ovarian cancer cells by directly targeting small family transcriptional repressor 1 (13).

The aim of the present study was to examine the expression of miRNA-148a-3p (miR-148a-3p) in ovarian cancer. We hypothesized that miR-148a-3p may suppress the metastasis of ovarian cancer primarily by targeting c-Met.

Materials and methods

Patients and tissue samples

Tissue samples were obtained from patients that underwent surgical resection at the Department of Obstetrics and Gynecology of Qilu Hospital (Jinan, China) between January 2012 and December 2013. In cases with histologically confirmed epithelial ovarian cancer, enrollment was limited to patients who underwent diagnostic procedures (e.g., paracentesis) only. Furthermore, any women with non-epithelial ovarian neoplasms, recurrent disease and other malignancies, or those who received neoadjuvant chemotherapy, were disqualified from the present study. The control group consisted of patients with benign ovarian tumor tissues. A total of 50 epithelial EOC (50.4±11.3 years; female) and 25 normal epithelial ovarian tissue sections (56.3±12.5 years; female) were obtained. No patients received chemotherapy or radiotherapy prior to surgery. Histopathological diagnoses were performed according to the criteria of the World Health Organization (14). All specimens were stored at −80°C for further use. The present study was approved by the Medical Ethics Committee of Shandong University (Shandong, China). Written informed consent was obtained from all patients prior to enrolment.

Cancer cell lines and culture

The SKOV3 and 293T cell lines used in the present study were supplied by the China Center for Type Culture Collection. EOC cells were incubated with Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 80 U/ml penicillin and 80 µg/ml streptomycin, at 37°C in a humidified atmosphere with 5% CO2 for 48 h.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from SKOV3 cells using TRIzol (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The quality of the RNA samples was assessed by determining the optical density (OD)260/OD280 ratio. TaqMan miRNA assays (Applied Biosystems, Thermo Fisher Scientific, Inc.) were used to analyze miR-148a-3p expression using the following specific primers: miR-148a-3p, 5-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAAA-3 and U6, 5-GTCGTATCCAGTGCAGGGTCCGGGTATTCGCACTGGATACGACAAATATG-3. U6 small nuclear RNA was used as a loading control and relative gene expression was calculated using the 2−∆∆Cq method (15). To measure the level of c-Met, cDNA was reverse-transcribed using a VigoScriptase kit (K009; Vigorous Biotechnology, Beijing, China). GAPDH was used as an internal control. The sequences of primers used for qPCR were as follows: miR-148a-3p, forward, 5′-GCTCAGTGCACTACAGAAC-3′; U6, forward, 5-GCGCGTCGTGAAGCGTTC-3; universal reverse primer, 5-GTGCAGGGTCCGAGGT-3; c-Met, 5-CAGGACTTGAAGCCAAGGGT-3 (forward) and 5-TGGGATGTTTCCCCGAGTTC-3 (reverse); GAPDH, 5-GAGAAGGCTGGGGCTCATTT-3 (forward) and 5-AGTGATGGCATGGACTGTGG-3 (reverse). PCR amplification was performed using 1 µg cDNA for the SYBR® Green Master mix (Invitrogen; Thermo Fisher Scientific, Inc.) using a Roche Lightcycler 480 (Roche Diagnostics, Indianapolis, IN, USA). The thermocycling conditions were as follows: 95°C for 10 min followed by 50 cycles of 95°C for 10 sec, 55°C for 10 sec, 72°C for 5 sec; 99°C for 1 sec; 59°C for 15 sec; 95°C for 1 sec; then cooling to 40°C.

Western blotting

Protein was extracted using a radioimmunoprecipitation assay buffer (Solarbio Science & Technology Co., Ltd., Beijing, China) from tissues and cells and was collected following centrifugation at 11,000 × g at 4°C for 20 min. A bicinchoninic protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to determine the protein concentration. A total of 20 µg of total protein lysate was separated using SDS-PAGE (10% gel) and then transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 8% nonfat dry milk suspended in PBST at 4°C overnight. The membranes were then incubated with primary antibodies against c-Met (cat. no. sc-64207; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or GAPDH (cat. no. sc-51631; 1:1,000; Santa Cruz Biotechnology, Inc.) at 4°C overnight. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated anti-immunoglobulin G (1:5,000; cat. no. ZB-2306; Zhongshan Gold Bridge Biological Technology Co., Beijing, China) for 2 h at room temperature and then washed followed by detection of protein bands using an enhanced chemiluminescent substrate (EMD Millipore). Primary antibodies against GAPDH were used as a control. The densitometric analysis of the bands was performed using ImageJ software (version 2.0; National Institutes of Health, Bethesda, MD, USA).

Oligonucleotide transfection

An miR-148a-3p inhibitor (sequence, 5′-ACAAAGTTCTGTAGTGCACTGA-3), miR-148a-3p mimic (sequence, 5′-TCAGTGCACTACAGAACTTTGT-3) and miRNA control (sequence, 5′-TTCTCCGAACGTGTCACGT-3), or siRNA targeting c-Met (sequence, 5′-AGAATGTCATTCTACATGAGC-3) and a non-target control siRNA (sequence, 5′-TTCTCTAGAACGTGTCAT-3) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). SKOV3 cells were transfected with the oligonucleotides at a final concentration of 20 nmol/l for 48 h using Hiperfect Transfection reagent (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol.

MTT assay

In brief, SKOV3 cells were seeded into a 96-well tissue culture plate at a density of 5×103 cells/well. Cells were cultured with medium only (containing 0.01% dimethyl sulfoxide as a negative control) or incubated with a miR-148a-3p mimic. Following incubation for 24, 48 and 72 h at 37°C, cell proliferation was determined using a MTT assay (Merck KGaA, Darmstadt, Germany). Following treatment, cells were cultured in fresh medium containing 0.5 mg/ml MTT for 4 h. Dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) was then added to the wells to dissolve the blue formazan products and the absorbance was measured at a wavelength of 450 nm using a plate reader. The experiments were performed in triplicate.

Transwell invasion assay

A Matrigel invasion assay was performed using a 24 well invasion chamber system (BD Biosciences, Franklin Lakes, NJ, USA) with a polycarbonic membrane. In brief, 1×106 SKOV3 cells were seeded in the upper chamber with 2 ml serum free DMEM culture and transfected with miR-148a-3p or si-c-Met, whereas fresh medium containing 10% FBS was plated in the lower chambers. Following incubation for 24 h at 37°C, cells on the upper chamber were stained using 0.5% crystal violet (dissolved in 10% acetic acid) at 37°C for 30 min. The invasive activity was determined by the measurement of the optical density at 560 nm. Cells were subsequently imaged under a light microscope (×40 magnification; Olympus Corporation, Tokyo, Japan). The results are presented as the mean of three separate experiments.

Prediction of miR-148a-3p-binding site

Putative miR-148a-3p-binding sites in the 3′ untranslated (UTR) MET mRNA region were predicted using the Target Scan program (http://www.targetscan.org). Position 1561–1567 of the MET 3′UTR was identified as a conserved binding site suitable for miR-148a-3p targeting.

Luciferase activity assay

293T cells were seeded into a 24 well plate at a density of 5×104 cells/well. Following incubation at 37°C for 24 h, a wild-type or mutated c-Met 3′-UTR luciferase reporter vector, pmir-GLO plasmid (Promega Corporation, Madison, WI, USA), combined with miR-148a-3p mimics or negative control (NC), were transfected into the cells at a final concentration of 20 nM using a Vigofect transfection reagent (Vigorous Biotechnology,) according to the manufacturer's protocol. Following 48 h transfection, relative luciferase units were determined using the Dual-Luciferase Reporter assay system (Promega Corporation, Madison, WI, USA). Renilla luciferase activity was used as the internal control.

Statistical analysis

Data are presented as the mean ± standard deviation. Differences between 2 groups were analyzed via the Student's t-test and differences among >2 groups were analyzed using one-way analysis of variance followed by post hoc Tukey analysis. Kaplan-Meier curves were created to investigate the association between the overall survival rate and miR-148a-3p expression and the differences between the survival curves were examined using the log-rank test. All statistical analyses were performed using SPSS (version 16.0: SPSS, Inc., Chicago, IL, USA) and P<0.05 was considered to indicate a statistically significant difference.

Results

miR-148a-3p expression is suppressed in ovarian cancer tissues

The expression of miR-148a-3p in ovarian cancer tissues was assessed. It was identified that miR-148a-3p expression was significantly decreased in EOC tissues compared with normal tissue (P<0.01; Fig. 1A). Furthermore, the prognostic value of miR-148a-3p in the overall survival of patients with EOC was recorded during a follow-up period of 60 months (follow-up data were obtained by telephone; Fig. 1B). Patients with EOC that exhibited miR-148a-3p levels below the median exhibited significantly poorer relapse intervals compared with patients that had miR-148a-3p levels above the median (Kaplan-Meier survival analysis; P=0.0052).

Figure 1.

miR-148a-3p expression is suppressed in ovarian cancer tissues. (A) Reverse transcription quantification polymerase chain reaction analysis of miR-148a-3p levels in ovarian cancer tissues compared with NCs. (B) KM survival analysis of patients with EOC exhibiting high (relative level of miR-148a-3p >2-fold) and low expression of miR-148a-3p (relative level of miR-148a-3p between 1- and 2-fold). **P<0.01. EOC, epithelial ovarian cancer; miR-148a-3p, microRNA-148a-3p; KM, Kaplan Meier; NC, normal controls.

Overexpression of miR-148a-3p inhibits the proliferation and invasion of SKOV3 cells

The effect of miR-148a-3p on SKOV3 cell proliferation and invasion was determined. Transfection with miR-148a-3p mimics significantly upregulated miR-148a-3p expression (P<0.001; Fig. 2A). In addition, the overexpression of miR-148a-3p, induced via transfection with miR-148a-3p mimics significantly suppressed the proliferation rate of SKOV3 cells (P<0.05; Fig. 2B). Furthermore, the overexpression of miR-148a-3p significantly decreased the invasiveness of SKOV3 cells compared with negative controls (P<0.001; Fig. 2C). These data indicate that miR-148a-3p suppresses proliferation and invasion of SKOV3 cells.

Figure 2.

Overexpression of miR-148a-3p inhibited the proliferation and invasion of SKOV3 cells. (A) Transfection with miR-148a-3p mimics significantly upregulated miR-148a-3p expression. (B) Overexpression of miR-148a-3p also significantly suppressed the SKOV3 cell proliferation rate. (C) The invasiveness of SKOV3 cells was decreased following miR-148a-3p overexpression compared with controls (magnification, ×40). *P<0.05; **P<0.01 and ***P<0.001 vs. NC. miR-148a-3p, microRNA-148a-3p; NC, negative controls; OD, optical density.

MET is a direct target gene of miR-144-3p

The possible target gene of miR-148a-3p was examined using TargetScan analysis, indicating the presence of a conserved binding site in the 3′UTR of c-Met (Fig. 3A). The results of the dual luciferase reporter assay demonstrated that miR-148a-3p significantly suppressed the relative luciferase activity of pmirGLO-c-Met-3′UTR compared with the blank vector (P<0.001; Fig. 3B). However, no changes of relative luciferase activity were identified in the mutated pmirGLO-c-Met-3′UTR. In addition, the expression of c-Met mRNA was significantly inhibited following transfection of miR-148a-3p mimics (P<0.01; Fig. 3C). Relative protein levels of c-Met were also significantly decreased when miR-148a-3p was overexpressed (P<0.01; Fig. 3D). These data indicate that c-Met is a direct target gene of miR-148a-3p.

Figure 3.

c-Met was identified as a direct target gene of miR-148a-3p. (A) A conserved binding site in the 3′UTR region of c-Met was identified using the TargetScan program. (B) The dual luciferase reporter assay demonstrated that miR-148a-3p significantly suppressed the relative luciferase activity of pmirGLO-c-Met-3′UTR compared with the blank vector. (C) mRNA levels of c-Met were significantly decreased following transfection of miR-148a-3p mimics. (D) Protein levels of c-Met were also decreased by miR-148a-3p overexpression. **P<0.01 and ***P<0.001 vs. NC. c-Met, tyrosine-protein kinase Met; UTR, untranslated region; miR-148a-3p, microRNA-148a-3p; RLU, relative luciferase units; NC, negative controls; Mut, mutated.

Decreased MET expression inhibits the malignancy of EOC

The expression of MET in ovarian cancer tissues was examined. mRNA levels of c-Met were significantly increased in EOC tissues compared with the control (P<0.001; Fig. 4A). To determine the effect of c-Met on EOC malignancy, a specific small interfering RNA targeting c-Met was selected (Fig. 4B). c-Met silencing decreased the proliferation rate of SKOV3 cells compared with scramble RNA (Fig. 4C). Similarly, the invasiveness of SKOV3 cells decreased when c-Met was silenced and was further decreased when miR-148a-3p was inhibited by transfection with an miR-148a-3p inhibitor for 48 h (P<0.001; Fig. 4D), indicating that the miR-148a-3p induced suppression of SKOV3 cells occurs primarily via c-Met.

Figure 4.

Decreased expression of MET inhibited the malignancy of EOC. (A) Levels of c-Met mRNA were significantly increased in EOC tissue. (B) Western blot analysis of siRNA targeting c-Met. (C) Silencing of c-Met decreased the proliferation of SKOV3 cells. (D) The invasiveness of EOC cells was also inhibited following the silencing of c-Met (magnification, ×40). **P<0.01 and ***P<0.001 vs. NC. MET, c-Met proto-oncogene; EOC, epithelial ovarian cancer; c-Met, tyrosine-protein kinase Met; NC, negative control; si-c-Met, siRNA targeting c-Met; OD, optical density; siRNA, small interfering RNA; 148i, miR-148a-3p inhibitor; si-c-Met+148i, siRNA targeting c-MET co-transfected with an miR-148a-3p inhibitor.

Discussion

Previous studies have demonstrated that the abnormal expression of certain miRNAs contributes to malignancy by inducing the proliferation, migration and invasion of cancer cells (16,17). In the present study, it was demonstrated that miR-148a-3p was downregulated in EOC tissues compared with normal controls. Furthermore, the overexpression of miR-148a-3p suppressed EOC cancer cell proliferation and invasion and c-Met was revealed to be a direct target of miR-148a-3p. Importantly, the inhibition of miR-148a-3p expression reversed the increase in SKOV3 cell invasion and proliferation induced by c-Met silencing. The results of the present study indicate that miR-148a-3p suppresses the development and progression of EOC cancer cells, primarily by targeting c-Met.

Ovarian cancer is one of the most common types of gynecological cancer (18,19). Among patients with ovarian cancer, ~90% have EOC subtypes. Due to the lack of effective early screening strategies (20) and a high rate of tumor relapse (21), mortality rates are high in patients with ovarian cancer. Epidemiological factors, genetic factors and molecular profiles represent the primary risk factors of EOC (22,23). Thus, it is crucial to develop more precise and effective treatment strategies to improve the survival rates of patients diagnosed with EOC.

The basic mechanism by which miRNAs regulate gene expression is primarily via the modulation of target genes. Thus, it is important to determine the possible target genes of EOC. The present study demonstrated that miR-148a-3p suppressed EOC proliferation and invasion primarily via c-Met. c-Met is an oncoprotein, which enhances tumorigenesis in a variety of different tumor types (8). c-Met also serves an important role in certain biological processes, including cell proliferation, migration and invasion (24). The aberrant expression of c-Met has been identified in various types of cancer, including gastric, bladder and colorectal cancer (25–27). The upregulation of c-Met is considered to be an independent predictor of poor patient prognosis (2,9). Furthermore, it has been demonstrated that c-Met increases the metastasis of uveal melanoma and liver cancer cells (28). In the present study, it was demonstrated that silencing c-Met significantly suppressed EOC cancer cell proliferation and migration. In addition, the inhibition of miR-148a-3p expression, combined with the silencing of c-Met, did not increase the invasiveness of SKOV3 cells. These data indicate that miR-148a-3p may act as a tumor suppressor in EOC by targeting c-Met.

In conclusion, the results of the present study demonstrated that miR-148a-3p expression was decreased in EOC cancer tissues and cell lines. In addition, the ectopic expression of miR-148a-3p suppressed SKOV3 cell proliferation and invasion. Furthermore, c-Met was identified as a potential target of miR-148a-3p, indicating that miR-148a-3p may serve as an EOC tumor suppressor primarily by targeting c-Met. However, due to the limited number of samples in the present study, further experiments are required to determine the use of miR-148a-3p as an effective biomarker in EOC.