The Mechanisms And Effects Of Maternally Expressed3, A Long Non-coding RNA, On The Proliferation Of Epithelial Ovarian Cancer Cells

Objective. Epithelial ovarian cancer (EOC) is one of the most common tumorsof the female reproductive system, and the mortality rate of this disease ranks firstamong gynecological malignant tumors. In spite of continuous efforts to improve thetherapeutic response, the prognosis for patients with EOC is still very poor, with anoverall5-year survival of approximately50%. Moreover, over70%of patients withEOC will relapse, although most advanced-stage patients undergo cytoreductivesurgery and platinum-based chemotherapy. Recently, several studies have revealedthat long non-coding RNAs (lncRNAs), which have lengths of>200nucleotides (nt),play important roles in tumor development. The maternally expressed3gene (MEG3)is an imprinted gene located on chromosome14q32that encodes an lncRNA—MEG3RNA. MEG3is expressed in many human tissues, such as ovary, testes, andbrain. However, recent studies have shown that expression of MEG3is lost inmultiple tumors including neuroblastomas, hepatocellular cancers, and gliomas and inmany cancer cell lines. Promoter methylation might account for the loss of expressionof MEG3in cancer. Studies have shown that promoter methylation plays a major role in silencing of the MEG3gene in clinically non-functioning adenomas (NFAs),meningiomas, and neuroblastoma cell lines. Moreover, the results of Zhou et alindicate that inactivation of the MEG3gene in these tumors can be attributed in partto promoter silencing by hypermethylation. MEG3may act as a tumor suppressorduring tumorigenesis. Overexpression of MEG3inhibits the growth of cancer cellslines such as MCF7, HeLa, HCT116, and IOMM-Lee and promotes apoptosis inHepG2, PLC/PRF/5, U251, and U87MG cells. Zhou et al determined that theantiproliferative activity of MEG3by regulating tumor suppressor p53. However, therelationship between MEG3and epithelial ovarian cancer (EOC) has not been studied,and the role of MEG3in EOC is still unknown. Therefore, on the basis of previousstudies, we hypothesized that MEG3may be a novel tumor suppressor gene in EOC.In this study, we determined whether or not MEG3RNA is downregulated in humanEOC and inhibits EOC proliferation via targeting of p53.Methods. Differences in the expression of MEG3and in the methylation statusof the MEG3promoter between EOC and normal ovary were analyzed by usingRT-PCR and methylation-specific PCR (MSP), respectively. MTT and EdU assaysand flow cytometric analysis were used to assess the growth of ovarian cancer cellsafter overexpression of MEG3. The target genes regulated by MEG3were detectedwith the Dual Luciferase Reporter system. The expression levels of target geneswere confirmed by using RT-PCR and western blot.Results. The expression of MEG3was downregulated in25%(5of20) orabsent in70%(14of20) of ovarian cancer tissues. In comparison,85%(17of20) ofnormal ovarian tissues expressed MEG3at high levels. The difference in expressionof MEG3RNA between normal samples and cancer samples was significant (P<.01).Furthermore, MEG3RNA was not detected in any of the ovarian cancer cell lines(OVCAR3, SKOV3, HP8910, or ES-2). An abnormally methylated pattern (M) ofthe MEG3differentially methylated region (DMR) was observed in60%(12of20)of patients with ovarian cancer who were evaluated. An unmethylated pattern (U)and a partially methylated pattern (M and U) were observed in15%and25%,respectively, of the20cancer samples. In comparison, most of the normal ovarian tissue samples (85%,17of20) displayed an unmethylated pattern (U). Thedifference in MEG3expression between normal and cancer samples was significant(P<.01) after using the chi-square test to correct for continuity. All of the ovariancancer samples that were categorized as “lost expression” displayed ahypermethylated pattern (M) while all of the normal samples that were categorizedas ‘‘high-expression’’ displayed an unmethylated pattern (U). Association analysisindicated that a significant negative correlation exists between the extent of CpGmethylation and the expression of MEG3RNA (χ2=32.73, r=-0.905, P<.01).Methylation in the4ovarian cancer cell lines (OVCAR3, SKOV3, HP8910, andES-2) was detected by MSP. This analysis revealed that all4of the cell lines wereabnormally methylated (M). Taken together, the data illustrate that partialdownregulation of MEG3is due to hypermethylation of the MEG3promoter and thatthe methylation status correlates with EOC grade. We treated human ovarian cancercells (OVCAR3cells) with different concentrations of5-aza-2-deoxycytidine(5-aza-CdR). Treatment with1uM,5uM,10uM, or20uM of5-aza-CdR resulted inre-expression of MEG3and partially reversed the abnormal methylation pattern ofthe MEG3promoter. These results indicate that CpG methylation may lead todownregulation of MEG3RNA in ovarian cancer cell lines. To investigate the role ofMEG3in cell growth, OVCAR3cells were transfected with pcDNA3.1-MEG3andthen subjected to proliferation and apoptosis assays. Results of the MTT assayshowed that proliferation was significantly inhibited in cells that overexpressedMEG3. In contrast to the NC and blank groups, the inhibition ratio of the MEG3group at12,24, and48hours after transfection was significantly different (37.26%,38.19%, and39.73%, respectively;**P<.01). An EdU assay showed that thepercentage of cells in S phase was reduced by35.01%±2.68%in OVCAR3cells thatoverexpress MEG3(**P<.01). The outcome of an analysis of the cell cycle indicatesthat OVCAR3cells were arrested at the G0/G1phase and that the percentage of cellsin the S and G2/M phases was also decreased. An apoptosis assay showed thatoverexpression of MEG3caused OVCAR3cells to undergo apoptosis. All of thedata presented above support the hypothesis that MEG3negatively regulates the growth of ovarian cancer cells. To understand the molecular mechanism by whichMEG3suppresses the growth of ovarian cancer cells, we investigated whetherMEG3regulates the activation of p53. OVCAR3cells were cotransfected withpGL3-p53and pcDNA3.1-MEG3. Figure6A shows that cells transfected withpcDNA3.1-MEG3had significantly increased levels of p53activity. Additionally, wealso found that overexpression of MEG3in OVCAR3cells increased p53, GDF15,and RB1mRNA and protein levels.Conclusions. Our data suggest that MEG3is epigenetically silenced in EOCdue to promoter hypermethylation, which may contribute to the development ofEOC.