Effect Of MicroRNA-10b On The Viability And Apoptosis Of Hepatocellular Carcinoma HepG2Cells

Aim:Investigate the impact of miRNA-10b in HepG2cells regarding promote-apoptoticprotein BIM and the anti-apoptotic protein MCL-1. Analysis the effect of miRNA-10bon the proliferation of HepG2cells. Try to explore the significance of miRNA-10b inthe proliferation, apoptosis and gene therapy of HCC.Methods:1.Establishing the transiently transfected cells: Use Liposome transfection method totransfect has-miRNA-10b and has-miRNA-10b-inhibitor eukaryotic expression vectorinto HepG2cells.2.Verifying the transfection efficiency: GFP expression was observed by fluorescencemicroscopy and miRNA-10b relative expression levels are measured by real-timePCR after transfection.3.Depicting cell growth curve and calculating the doubling time: Counting cellsnumber to depict growth curve and using the formula to Calculate the doubling time.4.Detecting the amount of the gene expression: Using real-time PCR to measure theexpression of BIM and MCL-1genes at the post-transcriptional mRNA levelsbetween experimental groups.5.Detecting the expression of protein: BIM and MCL-1protein levels are measuredby Western-blotting between the experimental groups.6.MTT assay: Using MTT assay to observe the cell growth and calculating the ratio ofliving cells in order to compare the proliferation of each group.7. Detecting apoptosis rate: Using AnnexinV-PI flow cytometry experiments to detectand compare apoptosis rate.Results：1.transfection efficiency is approximately50%-60%after transient transfection andmiRNA-10b expression among groups have significant difference.2.Depicting the cell growth curve of HepG2is aim to gain Basic growthcharacteristics of the cell lines. The doubling time of has-miRNA-10b group is theshortest and the Proliferation is markedly increased. While The doubling time ofhas-miRNA-10b-inhibitor group is the longest and the Proliferation is markedlydecreased. 3.The expression of mRNA and protein of BIM gene is negative correlation to theexpression of miRNA-10b. The MCL-1is on the contrary.4.MTT assay shows that the number of living cells of has-miRNA-10b group isincreased and cell proliferation speed up. The number and the speed ofhas-miRNA-10b-inhibitor group is lower.5.Flow cytometry shows that the rate of apoptosis of has-miRNA-10b-inhibitor is thehighest while the rate of has-miRNA-10b is the lowest.Conclusion：1.miRNA-10b eukaryotic expression vector can effectively regulate the expression ofmiRNA-10b in HepG2cells.2. High expression of miRNA-10b can inhibit the expression of BIM gene andpromote the expression of MCL-1. As a result, the cell is Excessive proliferation afterapoptosis dysregulated.3.Inhibiting miRNA-10b can slow down the cell proliferation and induce theapoptosis.