Study The Mechanism Of MiR-24-1-5P On Proliferation And Migration In Epithelial Ovarian Cancer

BackgroundOvarian cancer is one of the most popular gynecology malignant tumors. As its strong malignant behavior and tend to planting in abdomen, Ovarian cancer has the highest recurrence rate and mortality among gynecology malignant tumors. The p21-activated kinases 4 (PAK4) is serine/threonine protein kinases that plays crucial roles in cellular proliferation and invasion. MicroRNAs(miRNAs) are a new class of non-protein-coding, endogenous, small RNAs, and are known to negatively regulate gene expression or destroy the stability of genes via incomplete of complete matching with the 3’un-translational region(3’-UTR) of their target genes at the post-transcriptional level. They are involved in processes such as the regulation of organismal development, nerve differentiation, cell proliferation, apoptosis and fat metabolism. Our previous study found that the expression of miR-24-3p in epithelial ovarian cancer tissue is 20 fold of that in normal ovarian tissue, and PAK4 was the direct target site of miR-24-3p in TargetScan, which pushed us to the correlation study between miR-24, PAK4 and the epithelial ovarian cancer. Through miR-24 has three mature sequences, we focus on miR-24-1-5p in this thesis. We attend to set up human epithelial ovarian carcinoma cell line which was stable transfected with interference plasmids and to clarify the influence on ovarian cancer cell proliferation and migration, which provides the molecular mechanism in ovarian cancer, and indicates us a new direction of drug molecular targeted therapy for ovarian carcinoma.ObjectiveThe miR-24-1-5p interference plasmid was constructed and identified, and screened human epithelial ovarian carcinoma cells of stable transfection with miR-24-1-5p interference plasmid, to investigate the effect of miR-24-1-5p on the proliferation and migration in human epithelial ovarian carcinoma cell, which provides us with the experimental basis for clarifying the development and progress mechanism of the human epithelial ovarian cancer.Methods:1. pcDNA6.2-GW/EmGFP-miR-24-1-5p interference plasmid was constructed using BLOCK-iTTM Pol II miR RNAi Expression Vector Kits and sequenced by Sangon Biotech Company in Shanghai.2. pcDNA6.2-GW/EmGFP-miR-24-1-5p interference plasmid was transfected into human epithelial ovarian carcinoma cell line A2780 for 48 hours and screened through flow cytometry for fluorescence-activated cell sorting by GFP.3. The human epithelial ovarian carcinoma cells sorting by GFP were selected by the Positive Clone and Pressure Screen of Blasticidin for stable transfection cells.4. Western Blot was used to detect the expression of PAK4 in epithelial ovarian carcinoma cells which stably transfected with pcDNA6.2-GW/EmGFP-miR-24-1-5p interference plasmid.5. MTS assay was used to detect the effect of up-regulating miR-24-1-5p expression on epithelial ovarian carcinoma cell proliferation activity in vitro.6. Scratch test was applied to investigate the effect of up-regulating miR-24-1-5p expression on epithelial ovarian carcinoma cell migration.7. In vivo tumor growth assay was used to clarify effect of up-regulating miR-24-1-5p expression on epithelial ovarian carcinoma cell proliferation activity in vivo.Results:1. The sequencing data demonstrated that the miR-24-1-5p strand had been connected into the plasmid successfully.2. The transfection efficiency of epithelial ovarian carcinoma cells with pcDNA6.2-GW/EmGFP-miR-24-1-5p interference plasmid was about 16.0 percent detected by flow cytometry and gained the cells expressed with GFP.3. The epithelial ovarian carcinoma cells stably transfected with pcDNA6.2-GW/EmGFP-miR-24-1-5p interference plasmid were achieved by Positive Clone and Pressure Screen of Blasticidin at the concentration of 7μg/ml for 2 weeks.4. PAK4 in human epithelial ovarian carcinoma cell line with transfection of pcDNA6.2-GW/EmGFP-miR-24-1-5p interference plasmid was significantly decreased compared with that of transfection with the negative plasmids.5. After up-regulation of the expression of miR-24-1-5p in A2780 cells, cell proliferation viability was significantly enhanced than those of transfected with the negative plasmids in vitro (P<0.05).6. After up-regulation of the expression of miR-24-1-5p in A2780 cells, the migration viability of these cells was no significant difference with those of transfected with the negative plasmids (P> 0.05).7. After up-regulation of the expression of miR-24-1-5p in A2780 cells, cell viability of these cells was significantly enhanced than those of transfected with the negative plasmids in vivo (P<0.05).Conclusion:1. We successfully constructed the miR-24-1-5p interference plasmid and sequenced out stable transfection of human epithelial ovarian carcinoma cell line.2. MiR-24-1-5p inhibits the expression of PAK4 in the epithelial ovarian carcinoma cells.3. MiR-24-1-5p promotes the epithelial ovarian carcinoma cells proliferation both in vitro and vivo.4. MiR-24-1-5p has no effect on the migration ability of the epithelial ovarian carcinoma cells.