MicroRNA-519a inhibits the proliferation and promotes the apoptosis of ovarian cancer cells through targeting signal transducer and activator of transcription 3.

Ovarian cancer is a highly prevalent cancer among women. Recent studies have indicated that microRNAs (miRs) may serve important roles in the pathogenesis of ovarian cancer. miR-519a was observed to be downregulated in tissue samples of patients with ovarian cancer; however, its role in ovarian cancer requires further investigation. The aim of the present study was to examine the role of miR-519a in the pathogenesis of ovarian cancer and determine its direct target. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to examine the expression of miR-519a in 20 patients ovarian cancer and 20 normal ovarian tissue samples. Subsequently, SKOV3 cells were cultured and transfected with miR-519a mimics, while MTT and Annexin V assays were performed to investigate the role of miR-519a in the proliferation and apoptosis of SKOV3 cells. In addition, RT-qPCR and western blotting were used to determine the expression levels of miR-519a, signal transducer and activator of transcription 3 (STAT3), myeloid cell leukemia 1 (Mcl-1) and B-cell lymphoma-extra large (Bcl-xl) in untransfected and miR-519a mimic-transfected SKOV3 cells. Dual-luciferase reporter assay was also performed to confirm whether STAT3 was a direct target of miR-519a. The results revealed that miR-519a was significantly downregulated in tissue samples of patients with ovarian cancer as compared with the normal ovarian tissues. Furthermore, transient overexpression of miR-519a inhibited the proliferation and promoted the apoptosis of SKOV3 cells, as well as decreased the mRNA and protein expression levels of STAT3, Mcl-1 and Bcl-xl. Finally, dual-luciferase reporter assay confirmed that STAT3 was a direct target of miR-519a. In conclusion, the present study proved for the first time that miR-519a functions as a tumor suppressor by targeting STAT3 in ovarian cancer, suggesting that miR-519a may be a potential biomarker for the diagnosis and treatment of ovarian cancer.

Introduction

Ovarian cancer is one of the most common malignant tumors of the female genital organs, and its incidence rate (2.6/100,000 individuals) ranks third, following cervical and uterine cancer (1). However, the mortality rate of epithelial ovarian cancer is first among all types of gynecological tumors, posing a serious threat to the health of women (2). The common treatment methods of epithelial ovarian cancer include platinum-based cytotoxic chemotherapy and surgery (3). Although this treatment is effective for the majority patients with early-stage disease, a large number of women experience recurrence and chemoresistance, ultimately leading to disease-associated mortality (3,4).

Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT protein family. STAT3 mediates the expression of various genes in response to cell stimulation, serving a key role in various cell processes, including cell proliferation, apoptosis, survival, inflammation, invasion and metastasis (5,6). There has been increasing evidence suggesting that STAT3 is the most promising cell signaling target for chemoresistance in ovarian cancer (7–9).

MicroRNAs (miRNAs or miRs) are small non-coding RNA molecules found in animals, plants and certain viruses. Currently, miRNAs are receiving increasing attention as highly promising novel targets or tools in cancer therapy, since they serve important roles in the proliferation, progression and metastasis of solid tumors (10,11). In particular, miR-519a has been demonstrated to have a significant prognostic value in specific molecular glioblastoma subtypes, and to function as a tumor suppressor responsible for reductions in STAT3 expression and translation (12). Furthermore, in hepatocellular carcinoma cells, miR-519a promotes proliferation and inhibits apoptosis by targeting forkhead box F2 (13). However, the functional role of miR-519a in ovarian carcinoma, particularly in relation to STAT3 signaling, remains largely unknown.

Thus, the present study investigated whether miR-519a directly targeted STAT3, while the expression levels of STAT3, myeloid cell leukemia 1 (Mcl-1) and B-cell lymphoma-extra large (Bcl-xl) in ovarian cancer cells were analyzed. In addition, the clinical significance of miR-519a in ovarian cancer samples and its functional role in SKOV3 cells were examined.

Patients and methods

Patients and tissue samples

In total, 20 ovarian cancer and 20 adjacent normal ovarian tissue samples were obtained from stage III/IV ovarian cancer patients (34–55 years old) admitted to the Hebei General Hospital (Shijiazhuang, China) between April 2015 and April 2016, subsequent to obtaining informed consent. All patients were primary cases who had not received chemotherapy, radiotherapy or any other treatments prior to surgery. The samples were collected during surgery under sterile conditions. The present study was approved by the local ethics committee of Hebei General Hospital and conducted according to the Declaration of Helsinki (as revised in Tokyo, 2004).

Cell culture and transfection

Human ovarian cancer SKOV3 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in McCoy's 5A medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). Cells were seeded into 6-well plates and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. When cells reached 70% confluence cell transfection was performed using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cells were randomly divided into three groups as follows: Untreated cells (NC); cells transfected with empty vector (BL); and cells transfected with miR-519a mimics (Mimics; Genscript, Piscataway, NJ, USA).

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

Total RNA was isolated from the homogenized tissue samples or SKOV3 cells using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RNA concentration and quality were measured by using a NanoDrop™ 2000 instrument (Thermo Fisher Scientific, Inc.). miR-519a quantification was then conducted using the TaqMan MicroRNA Assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The relative expression of miR-519a was normalized to that of U6. For STAT3, Mcl-1 and Bcl-xl mRNA quantification, complementary DNA (cDNA) was synthesized from the total RNA using the RevertAid™ First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.). qPCR was then performed using the iTaq™ Universal SYBR-Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The mRNA expression levels of STAT3, Mcl-1 and Bcl-xl mRNA were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The thermocycling conditions were as follows: 95°C for 10 min (1 cycle), 95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec (40 cycles). The following primers were used in qPCR: miR-519a forward, 5′-ACACTCCAGCTGGGCTCTAGAGGGAAGCGC-3′ and reverse, 5′-ACACTCCAGCTGGGGTCCAGTTTTCCCAGGA-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; STAT3 forward, 5′-CAGCAGCTTGACACACGGTA-3′ and reverse 5′-AAACACCAAAGTGGCATGTGA-3′; Mcl-1 forward, 5′-TGCTTCGGAAACTGGACATCA-3′ and reverse, 5′-TAGCCACAAAGGCACCAAAAG-3′; Bcl-xl forward, 5′-CCCAGAGTTTGAGCCGAGTG-3′ and reverse, 5′-CCCATCCCTTCGTCGTCCT-3′; GAPDH forward, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ and reverse, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′. Triplicate Cq values were averaged and the relative expression levels were analyzed using the 2−ΔΔCq method (14).

MTT assay

The proliferation of SKOV3 cells was measured using MTT assay at 48 h after transfection. Briefly, the cells were plated onto 96-well plates (5×103 cells/well) and maintained at 37°C for 24 h. MTT solution (0.5 mg/ml) was then added to each well, and the cells were incubated for 3 h at 37°C. Subsequently, dimethyl sulfoxide was added to dissolve the formazan crystals. Cell proliferation was examined by measuring the absorbance at a wavelength of 490 nm using an ELISA plate reader (Bio-Rad Laboratories, Inc.).

Annexin V assay

In order to examine the cell apoptosis, an Annexin V assay was performed at 48 h after transfection using an Annexin-V-FLUOS Staining kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's protocol. Briefly, following centrifugation at 1,000 × g for 5 min at room temperature, the cells were resuspended in 500 µl binding buffer. Next, 5 µl Annexin V and 10 µl propidium iodide solution were added, and the cells were incubated for 15 min at room temperature in the dark. Flow cytometry was then performed with a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, USA) and the data was analyzed using CXP software version 1.0 (Beckman Coulter, Inc., Brea, CA, USA).

Western blotting

For the extraction of proteins, cells were placed in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) and centrifuged at 14,000 × g for 15 min at 4°C. Protein concentrations were then assayed using a NanoDrop 2000 instrument (Thermo Fisher Scientific, Inc.). Equal amounts of protein were separated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Subsequent to blocking with 5% skim milk in double-distilled water at room temperature for 1 h, the membranes were washed three times with phosphate-buffered saline containing 0.05% Tween (PBS-T) and incubated overnight at 4°C with primary antibodies directed against STAT3 (1:1,000; cat. no. sc-293151), Mcl-1 (1:1,000; cat. no. sc-12756), Bcl-xL (1:1,000; cat. no. sc-136207) and GAPDH (1:1,000; cat. no. sc-293335) (all Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The membranes were subsequently washed three times with PBS-T, followed by 1 h incubation with goat anti-mouse horseradish peroxidase-conjugated IgG secondary antibodies (1:10,000; cat. no. sc-516180; Santa Cruz Biotechnology, Inc.) at room temperature. Following incubation, the membranes were washed three times in PBS-T, and the antigen-antibody complexes were analyzed by enhanced chemiluminescence (Beyotime Institute of Biotechnology). The protein levels were normalized to GAPDH, which served as an internal control. The results were quantified by ImageJ software version 1.48 u (National Institutes of Health, Bethesda, MD, USA).

Dual-luciferase reporter assay

The targets of miR-519a were predicted using TargetScan (targetscan.org/vert_71). The complementary sequence of miR-519a was identified in the 3′-untranslated region (3′-UTR) of STAT3 mRNA. The cells were subsequently seeded into 24-well plates at 2.5×105 cells/well. The cells were then transfected with firefly luciferase reporter vectors (Sangon Biotech Co., Ltd., Shanghai, China) or control vectors containing Renilla luciferase (Sangon Biotech Co., Ltd.) using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). At 48 h following transfection, the cells in each group were measured using a dual-luciferase reporter assay system (Promega Corporation, Mannheim, Germany) according to the manufacturer's protocol. The luciferase activity was measured by using a luminometer. The results were obtained from three independent experiments performed in triplicate, and were normalized to the Renilla luciferase activity.

Statistical analysis

All data are expressed as the mean ± standard error. Statistical analysis was performed using SPSS software version 19.0 (IBM Corporation, Armonk, NY, USA) and GraphPad Prism version 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Differences among groups were analyzed by performing a one-way analysis of variance, followed by a Dunnett's post hoc test. P<0.05 was considered to indicate a difference that was statistically significant.

Results

Clinical implications of miR-519a expression in ovarian cancer tissues

To clarify the clinical implications of miR-519a in ovarian cancer, the miR-519a expression was analyzed by RT-qPCR in 20 ovarian cancer patients. The results demonstrated that the expression of miR-519a was significantly downregulated in ovarian cancer tissues as compared with the normal adjacent tissues (P<0.001; Fig. 1). These results indicated that miR-519a may serve an important role in ovarian cancer.

Figure 1.

Expression of miR-519a in ovarian cancer tissues compared with that in normal ovarian tissues (n=20 each). ***P<0.001. miR, microRNA.

miR-519a inhibits the proliferation and promotes the apoptosis of SKOV3 cells

MTT and Annexin V assays were performed to investigate the role of miR-519a in the proliferation and apoptosis of SKOV3 cells, respectively. Following the transfection of SKOV3 cells with miR-519a mimics, the data demonstrated a significant increase in miR-519a expression as compared with the negative control or blank groups, indicating that the transfection successfully induced miR-519a overexpression (P<0.001; Fig. 2A). Subsequently, an MTT assay was performed and revealed that miR-519a overexpression in SKOV3 cells resulted in a significantly inhibited proliferative ability (P<0.001; Fig. 2B). In addition, the Annexin V staining and flow cytometry assay also demonstrated that miR-519a overexpression in ovarian cancer SKOV3 cells resulted in significant promotion of apoptosis (P<0.001; Fig. 2C). These results verified that miR-519a was able to inhibit the proliferation and promote the apoptosis of SKOV3 cells in vitro.

Figure 2.

Overexpression of miR-519a reduced the proliferation and enhanced the apoptosis of SKOV3 cells. (A) Transfection with miR-519a mimics significantly increased the expression level of miR-519a in SKOV3 cells. (B) Cell proliferation, measured by MTT assay, was decreased following miR-519a overexpression in SKOV3 cells. (C) Cell apoptosis, measured by Annexin V staining and flow cytometric assay was increased following miR-519a overexpression in SKOV3 cells. ***P<0.001 vs BL group. miR, microRNA; NC, normal control; BL, blank control.

miR-519a inhibits the mRNA and protein expression levels of STAT3, Mcl-1 and Bcl-xl in SKOV3 cells

To further elucidate the mechanisms underlying the effects of miR-519a in SKOV3 cells, the mRNA and protein levels of STAT3, Mcl-1 and Bcl-xl were determined by RT-qPCR and western blotting, respectively. The results of RT-qPCR revealed that transient overexpression of miR-519a significantly reduced the expression levels of STAT3, Mcl-1 and Bcl-xl mRNA (P<0.001; Fig. 3A). Similarly, the results of western blotting identified that the levels of STAT3, Mcl-1 and Bcl-xl proteins were also significantly decreased (P<0.001; Fig. 3B). These results indicated that transient overexpression of miR-519a decreases the expression levels of STAT3, Mcl-1 and Bcl-xl.

Figure 3.

miR-519a inhibited the expression levels of STAT3, Mcl-1 and Bcl-xl in ovarian cancer SKOV3 cells. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blotting were performed to analyze the STAT3, Mcl-1 and Bcl-xl expression levels subsequent to miR-519a overexpression by mimic transfection of SKOV3 cells. Overexpression of miR-519a significantly reduced these mRNA and protein levels in the cells. ***P<0.001 vs BL group. miR, microRNA; NC, normal control; BL, blank control; STAT3, signal transducer and activator of transcription 3; Mcl-1, myeloid cell leukemia 1; Bcl-xl, B-cell lymphoma-extra large.

STAT3 is a direct target of miR-519a in SKOV3 cells

The complementary sequence of miR-519a was identified in the 3′-UTR of STAT3 mRNA (Fig. 4A), suggesting that miR-519a was able to bind to the 3′-UTR of STAT3′ mRNA. Subsequently, the study attempted to confirm whether the 3′-UTR of STAT3 mRNA directly interacted with the miR-519a as predicted, by conducting a dual-luciferase reporter assay. The results of this assay demonstrated that overexpression of miR-519a in SKOV3 cells significantly inhibited the dual-luciferase activity of STAT3 with the wild-type 3′-UTR (P<0.001; Fig. 4B). No evident effect was observed in the cells transfected with the mutant-type 3′-UTR. These results indicated that STAT3 is a direct target of miR-519a in SKOV3 cells.

Figure 4.

STAT3 was observed to be a direct target of miR-519a in SKOV3 cells. (A) The 3′-UTR of STAT3 mRNA was observed to contain the complementary sequence of miR-519a by bioinformatics analysis. (B) Overexpression of miR-519a significantly suppressed the dual-luciferase activity of the wt 3′-UTR of STAT3, but not of the mt 3′-UTR of STAT3. ***P<0.001. miR, microRNA; STAT3, signal transducer and activator of transcription 3; 3′-UTR, 3′-untranslated region; wt, wild-type; mt, mutant.

Discussion

Ovarian cancer accounts for the highest cancer-associated mortality among gynecologic cancer cases (2). Surgical debulking followed by platinum and taxane-based chemotherapy is the main treatment strategy for ovarian cancer (3). The improvement of surgery and chemotherapy strategies had little effect on the survival rate of the patients (15). Chemoresistance is one of the factors that lead to high morbidity and mortality in ovarian cancer, with the majority of patients succumbing to the disease due to chemoresistance (15). Downregulated expression of miR-134 has been reported to contribute to paclitaxel resistance in human ovarian cancer cells (16). In addition, miR-136 has been confirmed to enhance the antitumor effect of paclitaxel against the chemoresistance of ovarian cancer cells by targeting Notch3 (17).

miRNAs are currently identified as novel targets and tools in cancer therapy (10), and are known to serve important roles in ovarian cancer development, progression and drug resistance (18). In the present study, the roles of miR-519a in ovarian cancer were investigated. miR-519a has been reported to be a tumor suppressor in glioma by targeting the oncogenic STAT3 pathway (12). In the current study, the levels of miR-519a were found to be significantly downregulated in ovarian cancer tissues compared with those in normal adjacent tissues. The results of MTT assay and Annexin V assay also revealed that miR-519a inhibited the proliferation and promoted the apoptosis of SKOV3 cells.

The STAT3 signaling pathway has been confirmed as a therapeutic target in head and neck cancer (19), as well as in skin cancer (20). STAT3 is an important member of the STATs, it is stimulated by extracellular signals, including growth factors and cytokines, and it regulates cell proliferation, differentiation and apoptosis (21). In addition, Mcl-1 and Bcl-xl encode anti-apoptotic proteins and are members of the Bcl-2 family; thus, upregulation of Mcl-1 and Bcl-xl levels indicates tumor development. STAT3 regulates the expression of genes that represses apoptosis, including Mcl-1 and Bcl-xl (22,23). Overexpression of Mcl-1 and Bcl-xl is one of the key targets in the development of chemoresistance in cancer (24,25). In the present study, STAT3 was observed to be a direct target of miR-519a in SKOV3 cells. Subsequently, the expression levels of STAT3, Mcl-1 and Bcl-xl were detected in untransfected and miR-519a mimic-transfected SKOV3 cells. The results revealed that miR-519a overexpression inhibited the expression levels of STAT3, Mcl-1 and Bcl-xl.

In conclusion, the current study demonstrated that miR-519a directly targets STAT3, which functions as an oncogene in ovarian cancer. Decreased miR-519a expression was observed in ovarian cancer tissues. Furthermore, miR-519a inhibited the proliferation and promoted the apoptosis of ovarian cancer SKOV3 cells. Overexpression of miR-519a decreased the expression levels of STAT3, Mcl-1 and Bcl-xl. Therefore, these findings suggested that miR-519a may be a potentially effective therapeutic target for ovarian cancer in the future.