Introduction
Ovarian cancer is a common cancer type and is the fifth most frequent cause of cancer mortality in females (1,2). The majority of ovarian cancer patients are diagnosed in the advanced stages and the recurrence rate is >80% within two years (3). Despite the combination of surgery and chemotherapy, the five-year survival rate for ovarian cancer patients remains poor. Over the last three decades, this has increased from 37 to 45% (4). Therefore, a more effective therapeutic strategy is urgently needed and studies on the underlying molecular mechanisms of ovarian cancer may help improve the treatment.
The ovaries are important endocrine organs, secreting several types of estrogen, mainly 17β-estradiol (E2), which has been reported to play a critical role in ovarian cancer. The biological activity of E2 is mainly mediated by estrogen receptors (ERs) α and β. As a result, the dysregulation of ER may result in the development of ovarian cancer.
microRNAs (miRNAs) are a class of endogenous non-coding RNAs which can cause translation inhibition and degradation of their target mRNAs through binding to the target mRNA 3′ untranslated region (UTR). miRNAs play important roles in various biological processes, including cell proliferation, apoptosis, differentiation, migration and immune responses (5,6). Recently, a study has demonstrated that certain miRNAs have aberrant expression profiles in various cancer types (7). Furthermore, these miRNAs regulate specific critical oncogenes and tumor suppressors and are thus involved in tumorigenesis (8,9). An increasing number of studies on ovarian cancer have revealed that various miRNAs have oncogenic or anti-oncogenic roles, including miR-9, −335, −375 and −10b (10–13).
miR-206 has been reported to act as an important tumor suppressor in several cancers, including gastric, ovarian and colon cancer (14–16). Guo et al have previously reported that the expression of miR-206 is dysregulated in CD133+ ovarian cancer stem cells (17), indicating that miR-206 may play a role in ovarian cancer. Adams et al have identified ERα as a direct target of miR-206, which inhibits the mRNA and protein expression of ERα in human ovarian cancer cells (18). Furthermore, expression of miR-206 was shown to be associated with cellular proliferative inhibition and to impair invasion in ERα-positive endometrial carcinoma cells (19). However, the regulatory effect of miR-206 on ovarian cancer, as well as its relationship with ERα in ovarian cancer cells, remains to be studied.
The present study aimed to investigate the roles of miR-206 and ERα, as well as their regulatory patterns, in ovarian cancer cells.
Materials and methods
Tissue specimen collection
All protocols in the study were approved by the Ethics Committee of Xinxiang Medical University (Weihui, China). Informed consent was obtained from each patient in accordance with the guidelines of Xinxiang Medical University. In total, 21 primary ovarian cancer specimens and matched adjacent tissues were collected from patients at the Department of Gynecology and Obstetrics (First Affiliated Hospital of Xinxiang Medical University). Patients were diagnosed with primary ovarian cancer and were untreated, with no history of other tumors. All tissues were obtained following surgical removal and immediately snap-frozen in liquid nitrogen and stored at −80°C until use.
Reagents and materials
Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco-BRL (Carlsbad, CA, USA). Opti-MEM, fetal bovine serum (FBS), TRIzol, TaqMan qRT-PCR miRNA assay kit, RT-PCR kit, Lipofectamine 2000, miR-206 mimics and miR-206 inhibitor were purchased from Thermo Fisher Scientific (Waltham, MA, USA). MTT was purchased from Sigma (St. Louis, MO, USA). SYBR Green qPCR mix was purchased from TOYOBO (Osaka, Japan). Mouse anti-ERα, anti-matrix metalloproteinase (MMP)-2, anti-MMP9 and GAPDH monoclonal antibodies, along with rabbit anti-mouse secondary antibody and E2, were purchased from Abcam (Cambridge, UK). A 24-well transwell chamber was obtained from Corning Inc. (Corning, NY, USA). Matrigel was obtained from BD Biosciences (Franklin Lakes, NJ, USA).
Cell culture
Human ovarian cancer CAOV-3 and BG-1 cells lines were obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were maintained in the laboratory and cultured in DMEM containing 10% FBS at 37°C with 5% CO2.
RNA extraction and quantitative polymerase chain reaction (qPCR) analysis
Total cellular RNA was extracted using TRIzol agent. Following confirmation of the integrity of RNA, cDNA was synthesized from RNA using the RT-PCR kit in accordance with the manufacturer’s instructions. For the detection of miR-206 expression, TaqMan qPCR miRNA assay kit was used according to the manufacturer’s instructions and U6 was used as an endogenous control. For the detection of ERα mRNA expression, qPCR analysis was performed using SYBR Green qPCR mix and specific primers were obtained from Sangon Biotech (Shanghai, China). The following primers were used for amplification of ER α: sense, 5′-CCCACTCAACAGCGTGTCTC-3′ and antisense, 5′-CGTCGATTATCTGAATTTGGCCT-3′. GAPDH was used as an internal control: Sense 5′-ACAACTTTGGTATCGTGGAAGG-3′ and antisense, 5′-GCCATCACGCCACAGTTTC-3′. Independent experiments were repeated three times for each sample and the relative expression levels of genes were analyzed by use of the 2−ΔΔCt method.
Western blot analysis
Tissues or cells were solubilized in cold radioimmunoprecipitation lysis buffer. Next, protein (20 μg per lane) was separated with 10% SDS-PAGE and transferred from the gel to a nitrocellulose membrane. Membranes were blocked in 5% nonfat dried milk in phosphate-buffered saline (PBS)-Tween for 3 h and then incubated overnight with mouse anti-ERα, anti-MMP2, anti-MMP9 or anti-GAPDH monoclonal antibody (1:200, 1:100, 1:100 and 1:400, respectively). Following two washes for 5 min, the membrane was incubated with rabbit anti-mouse secondary antibody (1:20,000) for 40 min at room temperature. Next, immune complexes were detected using an enhanced chemiluminescence kit (Huyu Company, Shanghai, China). The membrane was scanned for the relative value of protein expression in gray scale by Image-Pro plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). The relative expression levels of protein were represented as the density ratio versus GAPDH.
Instruction of subgroup
In cell experiments, four subgroups were established. These were control, E2, E2 + miR-206 and E2 + miRNA negative control (NC) groups. In the E2 group, cells were treated with E2 at a concentration of 10 nM for 24 h, prior to MTT assay. In the E2 + miR-206 group, cells were transfected with miR-206 mimics using Lipofectamine 2000 according to the manufacturer’s instructions and treated with E2 at a concentration of 10 nM for 24 h. In the E2 + miRNA NC group, cells were transfected with non-specific miRNA using Lipofectamine 2000 for 24 h and subsequently treated with E2 at a concentration of 10 nM for 24 h.
MTT assay
For all groups, 10,000 cells per well were plated in a 96-well plate. Following treatment, the plates were incubated for 12, 24, 36 or 48 h at 37°C and 5% CO2. To assess cell proliferation, an MTT assay was performed according to the manufacturer’s instructions. In total, 50 μl MTT reagent (5 mg/ml) in PBS was added to each well and incubated for 4 h at 37°C and 5% CO2. Next, the supernatant was removed and 150 μl dimethyl sulfoxide was added. The absorbance was detected at 570 nm with a microplate reader (Bio-Rad, Hercules, CA, USA). Each assay was performed in triplicate wells and repeated three times.
Cell invasion assay
The cell invasion assays were performed in a 24-well transwell chamber which was precoated with 100 μg Matrigel. Cells in each group were collected and resuspended in serum-free DMEM at a concentration of 10,000 cells/ml. Next, 0.2 ml cell suspensions were added into the upper chamber and the bottom chamber was filled with 0.5 ml DMEM containing 10% FBS. Following incubation for 24 h at 37°C and 5% CO2, a cotton bud was used to remove the cells which had not passed through the polycarbonate membrane. Next, the cells that had passed through and adhered to the bottom were stained with trypan blue for 15 min and were subsequently photographed and counted.
Statistical analysis
Statistical analysis was performed using SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. The data were analyzed by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.
Results
Expression of miR-206 is downregulated in ERα-positive ovarian cancer tissues and two ERα-positive ovarian cancer cell lines
Firstly, the protein expression level of ERα was measured in 15 ovarian cancer tissue samples. As shown in Fig. 1A, 4 cases (nos. 7–9,15) showed high ERα protein expression and 6 cases (nos. 4–6,10,12,14) showed moderate ERα expression. These were grouped into the ERα-positive group. However, 4 cases (nos. 1–3,13) showed almost no expression of ERα and were grouped into the ERα-negative group.
To preliminarily investigate the role of miR-206 in ovarian cancer, qPCR was used to determine the miR-206 expression levels in ERα-positive and -negative human ovarian cancer tissues. As shown in Fig. 1B, miR-206 levels were much lower in the ERα-positive ovarian cancer tissues compared with those in ERα-negative tissues.
Next, the miR-206 expression levels were analyzed in the ERα-positive ovarian cancer lines, CAOV-3 and BG-1. Consistent with the aforementioned findings (20), CAOV-3 and BG-1 cells also showed a lower level of miR-206 compared with ERα-negative ovarian cancer tissues (Fig. 1C). These results indicate that miR-206 may play a role in ERα-positive ovarian cancer but not in ERα-negative ovarian cancer.
Introduction of miR-206 suppresses protein expression of ERα in CAOV-3 and BG-1 cells
It has been reported that ERα is directly targeted by miR-206. As a result, to further investigate the role of miR-206 and the association with ERα in ovarian cancer, CAOV-3 and BG-1 cells were transfected with miR-206 mimics. Non-specific miRNA mimics were used as an NC. Following transfection, the expression level of miR-206 in each group was determined. As shown in Fig. 2A, the expression level of miR-206 was significantly increased following transfection with miR-206 mimics, compared with that in the control and NC groups. These data suggest that miR-206 was successfully introduced into CAOV-3 and BG-1 cells.
Next, the mRNA and protein expression of ERα was measured. As shown in Fig. 2B, following transfection of CAOV-3 and BG-1 cells with miR-206 mimics, the mRNA levels of ERα were unchanged. However, ERα protein expression was significantly downregulated. As miRNA generally binds to the 3′ UTR of target mRNAs, results of this study indicate that miR-206 inhibits ERα expression at the post-transcriptional level.
miR-206 inhibits E2-induced cellular proliferation of ERα-positive ovarian cancer cells
The human ovarian cell lines, CAOV-3 and BG-1, have been shown to be ERα-positive and thus, ER-dependent (21,22). As a result, E2 was used to activate the ER-dependent signaling pathway for cellular growth. As hypothesized, treatment with E2 significantly upregulated cellular proliferation of CAOV-3 and BG-1 cells in a time-dependent manner (Fig. 3). miR-206 or non-specific miRNA mimics were further transfected into CAOV-3 and BG-1 cells, and it was found that the introduction of miR-206 mimics markedly inhibited E2-induced proliferation of CAOV-3 and BG-1 cells, while non-specific miRNA had no effect. Based on these findings, we hypothesize that miR-206 suppresses E2-induced cellular proliferation of ERα-positive ovarian cancer cells, by inhibiting the protein levels of ERα.
MiR-206 inhibits E2-induced cellular invasion of ERα-positive ovarian cancer cells
The ER-dependent signaling pathway has also been reported to be involved in cellular invasion of ER-dependent ovarian cancer cells (23). Thus, the effect of miR-206 on E2-induced invasion of CAOV-3 and BG-1 cells was analyzed. As shown in Fig. 4, treatment with E2 significantly promoted invasion of CAOV-3 and BG-1 cells. However, the introduction of miR-206 mimics effectively reversed it, while non-specific miRNA did not inhibit E2-stimulated cellular invasion. These data suggest that miR-206 downregulates E2-induced cellular invasion of ERα-positive ovarian cancer cells, in part, through inhibition of the protein expression of ERα.
It has been reported that E2 may increase mRNA and protein expression of MMP2 and MMP9 (24), which are key enzymes involved in cellular invasion (25). Thus, to further study the molecular mechanisms by which miR-206 suppresses E2-stimulated cell invasion in ERα-positive ovarian cancer cells, the ability of miR-206 to inhibit E2-induced upregulation of MMP2 and MMP9 was assessed. As shown in Fig. 4B, the expression levels of MMP2 and MMP9 were significantly upregulated in the E2 group compared with those in the control group. However, the introduction of miR-206 mimics effectively reversed this change and decreased the mRNA and protein expression of MMP9, while the introduction of non-specific miRNA did not. These findings suggest that MMP2 and MMP9 are downstream effectors of the miR-206-mediated inhibition of E2-induced invasion in ERα-positive ovarian cancer cells.
Discussion
To the best of our knowledge, the present study is the first to demonstrate downregulation of the expression of miR-206 in ERα-positive, but not ERα-negative ovarian cancer tissues. In addition, an inhibitory effect of miR-206 on ERα in ERα-positive ovarian cancer cell lines, OVCAR3 and SKOV3, was found. Furthermore, the introduction of miR-206 into ERα-positive ovarian cancer cells inhibited E2-induced cellular proliferation and invasion, at least in part via direct suppression of the expression of ERα.
Ovarian cancer is generally acknowledged as estrogen related. A meta-analysis of 2,500 patients revealed that 67% of primary ovarian cancers expressed ER which could be activated by E2, the main estrogen type secreted by the ovaries. E2 has been found to be involved in the etiology of ovarian cancer and to drive cellular proliferation in vitro and in vivo via ERα. Thus, E2 was used to activate the ERα-mediated cellular proliferation and invasion, and the therapeutic effect of miR-206 on E2-induced biological processes in ERα-positive ovarian cancer cells was explored.
Previous studies have reported that miR-206 is a tumor suppressor. Recently, a number of studies have demonstrated that the expression of miR-206 is downregulated in several cancer types, including gastric, breast, colon and laryngeal cancer, endometrioid adenocarcinoma and rhabdomyosarcoma (15–17,19,26,27). Yang et al previously found that the downregulation of miR-206 significantly correlates with tumor progression, and suggested that miR-206 is a potent prognostic marker of gastric cancer (14). Consistent with these findings, the present study supports the hypothesis that forced overexpression of miR-206 may effectively downregulate proliferation and invasion in ERα-positive ovarian cancer cells. In addition, these anti-proliferative and anti-invasion capacities may be explained by the direct inhibitory effect of miR-206 on ERα protein expression.
Li et al showed that miR-206 was downregulated in 93% of breast cancer tissues compared with matched normal adjacent tissues, indicating that miR-206 may be a novel prognostic marker for breast cancer (15). In addition, Chen et al suggested that forced expression of miR-206 may be associated with cellular proliferative inhibition and impaired invasion in ERα-positive endometrioid adenocarcinoma (19). Notably, like the ovaries, the mammary glands and endometrium also express high levels of ERα. Thus, based on results of previous studies and those of the present study, we hypothesize that there is a common regulatory mechanism involving miR-206 in organs expressing ERα at high levels.
The molecular mechanisms involved in the miR-206-mediated inhibition of E2-induced invasion in vitro were also studied, showing that MMP2 and MMP9 were downregulated by miR-206 in E2-treated OVCAR3 and SKOV3 cell lines. It is well established that MMP2 and MMP9 play crucial roles in the regulation of tumor invasion, metastasis, and angiogenesis (25,28). Merlo et al have demonstrated that E2 is able to increase the mRNA and protein expression of MMP2 and MMP9, as well as the levels of the active forms of the two enzymes released in the medium (24). However, no study to date has reported that miR-206 suppresses the expression of MMP2 and MMP9 stimulated by estrogen. Thus, the present study suggests, for the first time, that these two enzymes may act as downstream effectors of the miR-206-mediated inhibition of E2-induced invasion in ERα-positive ovarian cancer cells.
In conclusion, the present study has demonstrated that the expression level of miR-206 is significantly downregulated in ERα-positive human ovarian cancer tissues. In addition, introduction of miR-206 into ERα-positive ovarian cancer cells was shown to inhibit E2-induced cellular proliferation and invasion. Thus, these results indicate that miR-206 may be a promising candidate for endocrine therapy targeting ERα-positive human ovarian cancer.