A Research On The Roles Of MIR-21 And Its Regulative Effects On PTEN In Pathogenesis Of Ovarian Cancer

Objective:To investigate the expression of miR-21 and PTEN in epithelial ovarian cancers (EOC) and their correlation with the clinicopathologic features of the patients.Methods:Stem-loop real-time RT-PCR was used to detect the expression of miR-21 in epithelial ovarian cancers and benign epithelial ovarian cysts and normal ovarian tissues. PTEN protein was also examined with S-P immunohistochemistry method. Their correlation with histological differentiation, clinicopathological stage, and lymph node status was analyzed.Results:Among the 48 ovarian cancer samples analyzed, the relative expression of miR-21 (2-△△CT= 4.849±1.813) was significantly upregulated by 4-fold or more compared with the benign ovarian cysts (2-△△CT=1.133±0.291) and normal ovarian tissues (2-△△CT=1.057±0.126) (P＜0.01, respectively). No differences were observed in miR-21 relative expression between benign ovarian cysts and normal controls. The relative expressions of miR-21 were significantly higher in advanced stage and grade 3 tumor samples compared to the early stage and grade 1 to 2 tumor samples (2-△△CT=5.603±1.787 vs.3.341±0.254 and 2-△△CT=7.057±1.552 vs.3.845±0.680, p＜0.01 respectively). The same differences were observed between the lymph node positive group and negative group (2-△△CT= 7.095±1.728 vs.4.100±1.070, p＜0.01).20 of 48 (41.66%) ovarian cancer tissues were positive for PTEN, mainly weakly positive and expressed in the cytoplasm, which was distinctly lower than that in begin ovarian cysts and normal controls. Moreover, eight of 32 (25%) stageⅢ-Ⅳtissue samples were positive for PTEN, which was lower than that in stageⅠ-Ⅱtissue samples (12/16,75%). Three of 28 (10.71%) grade 3 tissue samples and two of 12 (16.66%) lymph node positive samples were positive for PTEN, which are obviously lower compared with gradel-2 samples (17/20,85%) and lymph node negative samples (18/36,50%) Both miR-21 and PTEN have no correlation with distinct histotypes of ovarian cancers.Seven cases were positive for PTEN among the 21 cases of ovarian cancer tissues with relatively low miR-21 expression (3.410±0.261), but only six cases were positive for PTEN among the 27 cases of ovarian cancer tissues with relatively high miR-21 expression (5.967±1.711). Spearman rank correlation analysis showed that PTEN expression was negatively correlated with miR-21 expression (r=-0.447, P＜0.01)Conclusion:Our data suggest that miR-21 aberrantly expressed in EOC and negtively correlated with PTEN, it might play an important role as an oncogene in the genesis and development of epithelial ovarian cancer. Objective:To detect the expression of PTEN and changes in cell proliferation, migration and invasion capabilities after downregulating the miR-21 expression in OVCAR3 cells with siRNA method. And investigate the mechanism by which miR-21 negatively controls the expression of PTEN, might play an important role as an oncogene, in the genesis and development of epithelial ovarian cancer.Methods:A short-hairpin RNA specifically targeting miR-21 was constructed and ligated with vector Linear pSIREN-RetroQ, resulting in three recombinants: pSIREN-miR-21-1,pSIREN-miR-21-2 and pSIREN-miR-21-Neg which were used to transfected OVCAR3 cells respectively. Stem-loop real-time RT-PCR was used to detect the expression of miR-21 and western blotting for PTEN protein. Cell proliferation, invasion and migration abilities were examined using MTT assay, Scratch-wound assay and transwell invasion assay respectively.Results:Seventy-two hours after infection, the infection efficiency reached more than 70% in the three groups of infected cells indicating successful transfection in all groups. miR-21 expression levels decreased and PTEN increased significantly both in pSIREN-miR-21-1 and pSIREN-miR-21-2 infected cells compared to pSIREN-miR-21-Neg infected cells and control group, that indicate inhibition of miR-21 can significantly increased PTEN expression in OVCAR3 cell lines. MTT assay indicates transfection of miR-21 inhibitor(pSIREN-miR-21-1 and-2) resulted in significant inhibition of 23.9% and 29.4% respectively at 72 hours in OVCAR3 cells indicating cell proliferation ability was decreased. Scratch-wound assay indicated cells migrated slowly after transfected with pSIREN-miR-21-1 and-2 compared with the control. Transwell invasion assay indicated the number of invasive cells in pSIREN-miR-21-1 group and group 2 was 40±3.56 and 29.5±2.38 that was significantly reduced relative to the control cells and pSIREN-miR-21-neg group (73±1.26,69±1.83).Conclusion:Our data suggest that the relative expression of miR-21 could be inhibited successfully using RNA interference. The downregulation of miR-21 could inhibite the cell proliferation, invasion and migration abilities through increasing the expression of PTEN protein in OVCAR3 cells that might play an important role in the genesis and development of epithelial ovarian cancer.