The Role Of MiR-135a And 17-β Estradiol In Epithelial Ovarian Cancer

Epithelial ovarian cancer (EOC) is not a single disease, but comprises a heterogeneous group of tumors. These tumors are classified according to their patterns of histological differentiation. The most common histological subtypes are serous adenocarcinomas, mucinous adenocarcinomas, endometrioid adenocarcinomas. EOC is often fatal because symptoms are usually absent in the early stage and nonspecific in advanced stages, so it is usually advanced by the time it is diagnosed. In order to better clarify the pathogenesis of EOC and ultimately identify novel therapeutic or diagnostic targets, it is vital to elucidate their molecular mechanisms. microRNAs (miRNAs) are small (20-24nt), endogenous, non-coding RNAsacting as novel post-transcriptional regulators of gene expression. With the discovery of miRNAs, a whole new level of gene regulation was revealed. These regulators mediate their effects by binding to 3'untranslated region (3'UTR) of their target genes through RNA-induced silencing complex (RISC) of miRNAs. In recent years, a growing body of literature has emerged identifying aberrant expression of miRNAs in tumorigenesis by regulating expression of the target oncogenes or tumor suppressor genes and a considerable number of miRNAs were predicted to display abnormal expression patterns. The miR-135 family encodes three miRNAs located on chromosomes 3p21.1, 12q23.1 and 1q32.1. Processes under the control of miR-135a include classic Hodgkin lymphoma (cHL), essential hypertension, human megakaryocytopoiesis, colorectal cancer, and bone and muscle phenotype development. However the role of miR-135a in epithelial ovarian cancer is still unclear.Aberrations of DNA methylation are now believed to be an important epigenetic alteration occurring early in many cancers including ovarian ones. In general, DNA methylation is one of the best-understood epigenetic changes in human cancers and may play important roles in carcinogenesis. Carcinogenesis is associated with changes in the epigenetic phenomenon, including two distinct and seemingly opposing trends: global decrease in cytosine methylation (hypomethylation) and methylation of cytocine in CpG islands (hypermethylation). This study found that down-regulation of HOXA10 in endometrial carcinoma is associated with promoter methlation. Also, estrogen plays an important role in regulation of HOXA10 expression in endometrial development, embryonic planting and breast cancer. We hypothesize that estrogen induces HOXA10 methylation, which in turn up-regulate HOXA10 expression in ovarian cancer.Through computer-aided algorithms, we obtained HOXA10 which is the predicted target gene of miR-135a. miR-135a mimics, miR-135a inhibitor and respective negative control were used to detect the relationship between miR-135a and HOXA10,Cell proliferation function was detected by MTT assay. The effect on apoptosis was detected by the expression of Caspase-3 and Bcl-2. Luciferase reporter assay was used to confirm the direct regulation by miR-135a of HOXA10. Finally we observed the effect of 17-βestradiol on the expression and promoter hypomethylation of HOXA10 gene. Better understanding of the mechanism of miR-135a and 17-βestradiol might shed light on developing therapeutic strategies. ObjectiveThis study was performed to detect the expression of miR-135a and identify the relationship between aberrant expression of miR-135a and the progression of epithelial ovarian cancer.MethodsRT-PCR and Real-Time PCR were used to examine the expression of miR-135a in 73 epithelial ovarian cancers and 55 matching controls.ResultsmiR-135a expression was significantly reduced in epithelial ovarian carcinoma (P < 0.01) and fell further with increasing FIGO stage and histological grade. Patients with high miR-135a expression had a longer progression-free survival (15+9.47 months) than the patients with low miR-135a expression (9.32+4.85 months) (P = 0.04). But there was no correlation between miR-135a expression and CA125 levels, lymph node metastases or residual tumour size.ConclusionsThis study shows a down-regulation of miR-135a in epithelial ovarian cancers which would associate with the disease development, suggesting miR-135a as a tumor suppressor gene. ObjectiveThe purpose of this study was to determine the relationship between miR-135a and HOXA10 expression, and to assess the effects of miR-135a on proliferation and apoptosis in epithelial ovarian cell lines.Methods1.Obtained the predicted target gene of miR-135a by computer-aided algorithms.2.miR-135a mimics, miR-135a inhibitor and respective negative control were tranfected into cells. RT-PCR, Real-Time PCR and Western Blotting were used to examine the expression of HOXA10 levels.3.Construct pMIR-REPORT Luciferase Plasmid containing 3'UTR of HOXA10.4.Luciferase reporter assay: Highly transfectable 293T cells transfected with pMIR-REPORT Luciferase construct,β-galactosidase control and PLemiR-135a or Non-silencing lentiviral miRNA control per well. Forty-eight hours later, luciferase activity was measured.5.Cell proliferation function was detected by MTT assay. The effect of miR-135a in apoptosis was detected by the expression of Caspase-3 and Bcl-2.Results1.RT-PCR: Although miR-135a did not significantly decrease HOXA10 expression at 24 h, varying degrees of reduction were found in the other two times. The Real-Time PCR assay further demonstrated the inverse relationship between miR-135a and HOXA10, after 48 h and 72 h, the Real-Time PCR showed a significant reduction of HOXA10 expression in all three miR-135a-treated cell lines compared with matching control (P < 0.05). Western Blotting analysis of these cells confirmed this result.2.As expected, analysis of HOXA10 levels at 24 h, 48 h and 72 h after miR-135a inhibitor transfection showed an up-regulation (P < 0.05). 3.The luciferase activity for pMIR-REPORT Luciferase vector containing HOXA10 3'UTR was decreased 67.8% at 48 h in the PLemiR-135a-transfected 293T cells compared with Non-silencing lentiviral miRNA control (P < 0.01).4.Although we did not find any difference in proliferation of all three cell lines after miR-135a mimics transfection for 24 h (P > 0.05), a significant decrease in proliferation was observed at 48 and 72 h compared with the control (P < 0.05). Dissimilarly, during the first 2 days after miR-135a inhibitor transfection, there was no difference between control and experiment groups (P > 0.05), however, after 72 hours, the miR-135a inhibitor significantly increased cell proliferation (P < 0.003).5.The Caspase-3 activity was significantly higher 48 hours after transfected with miR-135a mimics than in cell lines transfected with control vector (P < 0.05). Accordingly, an inverse result was found in miR-135a inhibitor transfected cells (P < 0.05). Furthermore, we confirmed the down-regulation of Bcl-2 levels by miR-135a mimics and an adverse outcome by miR-135a inhibitor.ConclusionsmiR-135a affect cell proliferation and apoptosis by targeting 3'UTR of HOXA10.Partâ…¢The effect of 17-βestradiol on the expression and promoter hypomethylation of HOXA10ObjectiveTo investigate the effect of 17-βestradiol on the expression and promoter hypomethylation of HOXA10 gene in human ovarian adenocarcinoma cell line.Methods1.The ovarian adenocarcinoma cell line (SKOV3) was incubated with 1×10^-6, 1×10^-7 or 1×10^-8mol/L 17-βE₂ for 48 h. Then the expression of HOXA10 mRNA and protein were detected by Real-Time PCR and Western Blotting2.SKOV3 genetic DNA of each group was extracted, purified and modified. DNA methylation status was detected by methylation specific polymerase chain reaction (MSP).Results1.Compared with control, HOXA10 mRNA and protein levels in the SKOV3 cell line were increased by 17-βE₂ treatment in a dose-dependent manner (P < 0.05).2.After 48 hours long exposure to 17-βE₂, HOXA10 promoter region showed partial methylation.Conclusions17-βE₂ could demethylate the promoter of HOXA10, and up-regulated HOXA10 expression in human ovarian adenocarcinoma cell line SKOV3.