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MicroRNA-199a-3p inhibits ovarian cancer cell viability by targeting the oncogene YAP1

MicroRNA-199a-3p (miR-199a-3p) is aberrantly expressed in various types of cancer where it exhibits a tumor suppressive role. However, the biological role of miR-199a-3p in ovarian cancer (OC) remains unclear. The present study aimed to investigate whether miR-199a-3p was a tumor suppressor in OC and to identify the possible mechanisms. It was found that miR-199a-3p expression was significantly downregulated in the tumor tissues and blood samples of patients with OC, as well as in three OC cell lines. In addition, its low expression was closely associated with International Federation of Gynecology and Obstetrics disease stage, histological grade and lymph node metastasis. It was demonstrated that overexpression of miR-199a-3p inhibited the viability and promoted apoptosis of OV90 and SKOV-3 cells. In addition, Yes-associated protein 1 (YAP1), a well-known oncogene, was identified as a direct target of miR-199a-3p in OC cells. Additionally, it was observed that YAP1 was significantly increased and inversely correlated with miR-199a-3p expression in OC tissues. Notably, YAP1 overexpression abrogated the tumor suppressive effects of miR-199a-3p in vitro. Collectively, the present results indicated that miR-199a-3p suppressed viability in OC cells, at least partly via inhibiting the YAP1 oncogene, suggesting that miR-199a-3p may act as a biomarker and therapeutic target for patients with OC.


Introduction
Ovarian cancer (OC) is a malignant tumor that seriously threatens women's health worldwide (1). In 2012, OC was one of the most frequent cancer diagnoses worldwide, with 238,700 new cases, and one of the leading causes of cancer mortality, with 151,900 deaths (2). Despite advances in conventional therapies, such as surgical treatment, radiotherapy and chemotherapy, the 5-year survival rate of patients with OC is <42.9% due to the high occurrence of therapy resistance and metastasis (3,4). Thus, it is urgently required to explore new strategies for the early detection of OC in patients and to discover new therapeutic targets for the treatment of OC.
MicroRNAs (miRNAs/miRs) are a family of short, small, non-coding RNAs with a length of ~22 nucleotides, which negatively regulate target gene expression through either translation repression or RNA degradation (5,6). Increasing evidence demonstrates that various miRNAs are aberrantly expressed in OC tissues and are involved in several pathophysiological processes, including cell proliferation, apoptosis and invasion (7,8). Various studies highlight the tumor suppressive role of miR-199a-3p in different types of cancer. For example, miR-199a-3p decreases esophageal cancer cell proliferation through repression of p21 activated kinase 4 (9). Liu et al (10) demonstrated that overexpression of miR-199a-3p inhibits cell proliferation, migration and invasion in clear cell renal cell carcinoma. Guan et al (11) revealed that miR-199a-3p can effectively inhibit tumorigenesis of xenografts in nude mice by regulating zinc-fingers and homeoboxes 1-dependent p53 upregulated modulator of apoptosis signals. Notably, several studies demonstrate the tumor suppressive role of miR-199a-3p in OC. For instance, Cui et al (12) showed that miR-199a-3p enhances cisplatin sensitivity of ovarian cancer cells by targeting integrin β8. Deng et al (13) revealed that overexpression of miR-199a-3p impairs the migratory, invasive and tumorigenic capabilities of ovarian cancer cells by inhibiting discoidin domain receptor tyrosine kinase 1 expression. However, the underling mechanisms of miR-199a-3p in OC remain to be fully elucidated.
The present study investigated the expression pattern of miR-199a-3p in OC tissue and cells. In vitro experiments were performed to explore the functional role of miR-199a-3p in OC cells and the underlying mechanisms. The present findings may provide a new insight that presents tentative strategies for the diagnosis and therapy of OC.  The expression levels of miR-199a-3p and YAP1 were normalized to those of of U6 and GAPDH, respectively. RT-qPCR assays were performed in triplicate and the relative expression of each gene was calculated using the 2 -∆∆Cq method (15).  Caspase-3 activity. Following transfection, OV90 and SKOV-3 cells were harvested and caspase-3 activity was measured using a Caspase-3 Activity kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The optical density was then detected using a microplate reader (Model 680, Bio-Rad Laboratories, Inc.) at an absorbance of 405 nm.

MicroRNA-199a-3p inhibits ovarian cancer cell viability by targeting the oncogene YAP1
Immunofluorescence assay (IFA). Following transfection, OV90 and SKOV-3 cells were fixed in absolute ethyl alcohol for 30 min at room temperature. After washing twice with PBS, the cells were fixed with 4% paraformaldehyde for 10 min and blocked for 2 h at 37˚C with 5% skimmed milk in PBST.
Then the fixed cells were stained with a primary antibody against cleaved-caspase-3 (1:1,000; cat. no. 9664; Cell Signaling Technology, Inc.) for 1 h at room temperature. Subsequently, a secondary antibody conjugated with FITC (1:100; cat. no. F1763; Sigma-Aldrich; Merck KGaA) was added for 2 h in the dark at room temperature, Fluorescence images were captured using an inverted fluorescence microscope (x200 magnification).
Western blot analysis. Western blot was performed as previously described (16). Briefly, total protein was extracted from cells using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) and the protein concentration was measured using a Bicinchoninic Acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). 40 µg protein was separated via 15% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (EMD Millipore). The membrane was blocked with 5% skimmed milk for 2 h at 4˚C overnight and probed with primary antibodies as follows: YAP1 (1:1,000; cat. no. 14074) and β-actin (1:2,000, cat. no. 4970) at 4˚C overnight. Membranes were subsequently incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000; cat. no. 7074) for 1 h at room temperature. All antibodies were obtained from Cell Signaling Technology, Inc. Proteins bands were visualized using an enhanced chemiluminescence detection system (Cytiva). The protein bands were developed using an ECL kit (Cytiva) and blot bands were quantified using ImageJ (version 1.46; National Institutes of Health).
Statistical analysis. Statistical calculations were performed using SPSS 13.0 software (SPSS, Inc.). Unpaired t-test was used for intergroup comparisons. Continuous data from multiple groups were analyzed using one-way ANOVA, followed by Tukey's post hoc test. All data are presented as the mean ± standard deviation. Spearman's correlation analysis was used in correlation analysis. All 50 patients with OC were divided into the high miR-199a-3p or low miR-199a-3p expression group, according to the median fold-change values. Chi-square test was performed to assess the association between serum miR-199a-3p expression levels and clinical variables. P<0.05 was considered to indicate a statistically significant difference.

miR-199a-3p expression is downregulated in OC.
To explore the role of miR-199a-3p in OC, the expression levels of miR-199a-3p in 50 paired OC and matched adjacent non-tumor tissues were first analyzed via RT-qPCR. As shown in Fig. 1A,   compared with adjacent non-tumor tissues, the expression levels of miR-199a-3p were significantly downregulated in OC tissues. Additionally, miR-199a-3p expression in 50 peripheral blood samples from patients with OC and 50 peripheral blood samples from healthy donors (normal group) were examined. Compared with the normal group, the expression levels of miR-199a-3p were also decreased in peripheral blood samples from patients with OC (Fig. 1B). To validate whether downregulation of miR-199a-3p was also present in OC cell lines, the expression levels of miR-199a-3p in several OC cell lines (TOV112D, OV90 and SKOV-3) were detected, with the normal cervical epithelial IOSE80 cells acting as a control. It was observed that miR-199a-3p expression was significantly decreased in OC cell lines compared with in IOSE80 cells (Fig. 1C). Next, the association between miR-199a-3p expression levels and the clinical characteristics of patients with OC was further investigated. All 50 cases of patients with OC were divided into the high miR-199a-3p expression group and low miR-199a-3p expression group, according to the median fold-change values. As demonstrated in Table I, miR-199a-3p expression was significantly associated with International Federation of Gynecology and Obstetrics (FIGO) disease stage, Grade and lymph node metastasis (17); however, no statistically significant association between miR-199a-3p expression and age, residual disease or ascites was identified. The present findings suggested that miR-199a-3p may be a potential marker for the diagnosis and prognosis of patients with OC.

miR-199a-3p overexpression inhibits cell viability and promotes apoptosis.
To further examine the effect of miR-199a-3p on OC, miR-199a-3p mimics were added to the cultured OV90 and SKOV-3 OC cell lines as they exhibited the lowest expression of miR-199a-3p. After 48 h, RT-qPCR analysis demonstrated that miR-199a-3p expression was significantly increased following miR-199a-3p mimics transfection ( Fig. 2A). The results of the CCK-8 assay revealed that miR-199a-3p overexpression significantly suppressed the viability of OV90 and SKOV-3 cells compared with in the mimics NC group (Fig. 2B and C). Additionally, miR-199a-3p overexpression resulted in a significant increase in caspase-3 activity and cleaved-caspase-3 expression in OV90 and SKOV-3 cells, as determined by caspase-3 activity assay and IFA ( Fig. 2D and E). The percentage of apoptotic cells in miR-199a-3p mimics-transfected OV90 and SKOV-3 cells was evaluated by flow cytometry. As demonstrated in Fig. 2F, the apoptotic rate was significantly increased compared with mimics NC group. Overall, the current results revealed that overexpression of miR-199a-3p inhibited cell viability by inducing apoptosis.

YAP1 is a direct target of miR-199a-3p in OC cells.
To further characterize the molecular mechanisms involved in the tumor-suppressive role of miR-199a-3p in OC cell viability, potential target genes of miR-199a-3p were searched for using two publicly available databases TargetScan 7.0 (targetscan. org/) and miRanda (microrna.org/). Through bioinformatics prediction, a putative target site of miR-199a-3p was found in the 3'-untranslated region (UTR) of YAP1 mRNA (Fig. 3A). First, it was established that miR-199a-3p expression was significantly decreased in OC cells following transfection with the miR-199a-3p inhibitor (Fig. 3B). To experimentally validate the possibility that YAP1 was targeted by miR-199a-3p, luciferase reporter assay was performed. The results demonstrated that the miR-199a-3p mimics significantly inhibited the luciferase activity using the YAP1-3'-UTR wild type (wt) reporter, while the miR-199a-3p inhibitor caused an increase in luciferase activity; however, no changes were observed using the YAP1 3'-UTR mutant (mut) reporter with miR-199a-3p mimics or inhibitor (Fig. 3C). Western blot analysis confirmed that miR-199a-3p overexpression significantly inhibited YAP1 expression in OV90 and SKOV-3 cells at protein level (Fig. 3D). In addition, YAP1 expression was detected in 50 paired OC and matched adjacent non-tumor tissues by RT-qPCR, and the results showed that YAP1 expression was significantly increased in OC tissues compared with that in adjacent normal tissues (Fig. 3E). Further correlation analysis indicated that YAP1 expression was inversely correlated with miR-199a-3p expression in OC tissues (r=-0.7648; P<0.01; Fig. 3F). The present results indicated that miR-199a-3p directly targeted YAP1 and suppressed its translation in OC.
Overexpression of YAP1 reverses the antitumor effect of miR-199a-3p in OC cells. To ascertain whether YAP1 may be involved in the antitumor effects of miR-199a-3p, pcDNA-YAP1 and miR-199a-3p mimics were co-transfected into OV90 and SKOV-3 cells. As expected, the protein expression levels of YAP1 were significantly increased in OV90 and SKOV-3 cells following pcDNA-YAP1 transfection, as determined by western blot analysis (Fig. 4A). Subsequently, the cell viability and the activity of caspase-3 were evaluated. As shown in Fig. 4B, overexpression of YAP1 significantly attenuated the inhibitory effect of miR-199a-3p overexpression on cell viability of OV90 and SKOV-3 cells. Additionally, it was shown that the activity of caspase-3 induced by miR-199a-3p mimics was reversed by YAP1 overexpression (Fig. 4C). The effect of YAP1 on miR-199a-3p-mediated cell apoptosis was further analyzed by flow cytometry, and the results showed that YAP1 overexpression significantly suppressed apoptosis compared with that in the miR-199a-3p mimics group (Fig. 4D). The current results suggested that miR-199a-3p may function as a tumor suppressor through suppressing the oncogene YAP1.

Discussion
The present study identified that miR-199a-3p expression was downregulated in OC tissues and cell lines. miR-199a-3p expression was associated with FIGO disease stage, grade and lymph node metastasis. In addition, overexpression of miR-199a-3p inhibited OC cell proliferation and promoted apoptosis via targeting the oncogene YAP1. Therefore, miR-199a may be a potential therapeutic target for the treatment of patients with OC.
Previous studies showed that miR-199a-3p is frequently downregulated in several types of human cancer and generally has a tumor suppressive role (18,19). For example, miR-199a-3p suppresses tumor growth, migration, invasion and angiogenesis in HCC by targeting vascular endothelial growth factor A and its receptors, hepatocyte growth factor inhibitor NC in OV90 cells. (C) Luciferase assay of 293T cells co-transfected with firefly luciferase constructs containing the YAP1 wt or mut 3'-UTRs and miR-199a-3p mimics, mimics NC, miR-199a-3p inhibitor or inhibitor NC, as indicated (n=3). Data are presented as the mean ± standard deviation (n=3) of one representative experiment. ** P<0.01 vs. mimics NC; ## P<0.01 vs. inhibitor NC. (D) OV90 and SKOV-3 cells were transfected with miR-199a-3p mimics and mimics NC for 48 h and the protein expression levels of YAP1 were determined by western blotting. Data are presented as the mean ± standard deviation (n=3) of one representative experiment. ** P<0.01 vs. mimics NC. (E) YAP1 expression was measured by RT-qPCR in 50 pairs of OC and matched adjacent non-tumor tissues. ** P<0.01 vs. adjacent tissues. (F) Spearman's correlation analysis was used to analyze the correlation between YAP1 and miR-199a-3p expression in OC tissues. YAP1, Yes-associated protein 1; UTR, untranslated region; miR, microRNA; wt, wild type; mut, mutant; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR. and matrix metallopeptidase 2 (19). In addition, restoration of miR-199a-3p decreases the invasiveness of HCC cell lines by controlling the expression of the mechanistic target of rapamycin (20). Notably, Kinose et al (21) identified that miR-199a-3p expression is downregulated under hypoxia and that the restoration of this miRNA significantly inhibits OC progression. Although miR-199a-3p has been reported to function as a tumor suppressor in OC (12), the role and precise mechanisms in OC remain to be further investigated. In the present study, a significant downregulation of miR-199a-3p expression in OC tissues and in peripheral blood samples from patients with OC was observed. Furthermore, miR-199a expression was significantly associated with FIGO disease stage, grade and lymph node metastasis, suggesting that its downregulation may contribute to the malignant progression of OC. It was identified that miR-199a overexpression significantly inhibited OC cell viability and promoted apoptosis, consolidating the functional roles of miR-199a in OC.
Emerging evidence shows that miRNAs exist in cells, as well as in circulating blood, reflecting tissue or organ conditions (22). miRNAs generated in the cytoplasm can not only affect the function of the cell in which they are produced, but they can also be released into the blood stream and are taken up to regulate the gene expression of distant target cells (23).
Häusler et al (24) investigated the whole blood-derived miR profiles of patients with OC and suggested that miRNAs possess potential as minimally invasive diagnostic markers. Kan et al (25) identified that miR-200a, miR-200b and miR-200c are significantly elevated in the serum of patients with OC and suggested that their presence may be used as a predictor of OC. The aforementioned studies support the idea that the detection of OC-associated miRNAs from the peripheral blood may become a valuable method for the early diagnosis of this disease in future clinical practice. For miR-199a-3p, several studies reported its abnormal expression in different types of human cancer. For example, Chai et al (26) identified that plasma miR-199a-3p expression is significantly lower in patients with glioma. Nonaka et al (27) reported that miR-199a-3p expression is significantly decreased in the post-operative serum from patients with colorectal cancer. All these results suggest that miR-199a-3p may be used as a promising novel biomarker for the diagnosis and prognosis of cancer. The present study identified that miR-199a-3p expression was downregulated in peripheral blood samples from patients with OC, suggesting that miR-199a-3p may have the potential as a diagnostic biomarker in OC.
YAP1 is suggested to be a potent oncogene and its expression is found to be elevated in several types of cancer (28-31). For example, Liu et al (32) showed that overexpression of YAP1 promotes invasion, migration and viability in colon cancer cells. Sun et al (33) reported that YAP1 expression is increased in gastric cancer tissues and overexpression of YAP1 promotes cell proliferation and invasion in vitro and in vivo. Zhou et al (34) revealed that YAP1 is highly expressed in nasopharyngeal carcinoma (NPC) cells and that YAP1-knockdown suppresses the proliferation, migration and invasion of NPC cells. Notably, several studies reported the oncogenic role of YAP1 in OC. For example, Jeong et al (35) demonstrated that the activation of YAP1 is significantly associated with the prognosis of patients with OC. Cho et al (36) reported that YAP1 expression is higher in OC compared with normal control samples and that high YAP1 expression may be associated with a poor overall survival in patients with OC. However, YAP1 expression and its correlation with miR-199a-3p in OC have rarely been reported. Using bioinformatics tools, the present study revealed that miR-199a targeted YAP1 and provided evidence that YAP1 was modulated by miR-199a. In addition, an inverse correlation was identified between YAP1 and miR-199a-3p expression, and YAP1 overexpression abrogated the antitumor effect of miR-199a in OC cells. The current results indicated that miR-199a-3p may regulate YAP1 expression to modulate the proliferation and apoptosis of OC cells.
However, there are some limitations to the present study. Due to the limitation in experimental conditions and funds, further research is required to investigate the expression levels of miR-199a-3p in more clinical samples. Furthermore, other targets of miR-199a-3p should be identified in future studies.
In conclusion, the present study demonstrated that miR-199a-3p expression was downregulated in peripheral blood samples from patients with OC, suggesting that miR-199a-3p may have the potential as a diagnostic biomarker in OC. The results further demonstrated that miR-199a-3p suppressed the proliferation and promoted apoptosis of OC cells by directly targeting YAP1. Based on these findings, it is proposed that the miR-199a-3p/YAP1 axis may serve as a novel biomarker for new targets for OC therapy in the future.