Study On The Role And Mechanism Of TOP2A In Hepatocellular Carcinoma Cells

Objective:To study the biological function of TOP2A in hepatocellular carcinoma cells,and to further explore the possible molecular mechanism of TOP2A on hepatocellular carcinoma cells by RNA transcriptome sequencing analysis of Huh7 cells with TOP2A knockdown,and to provide theoretical basis for the analysis of the mechanism of TOP2A in hepatocellular carcinoma.Methods:The expression of TOP2A in liver cancer cells and normal liver epithelial cells was detected by RT-QPCR,and the expression of TOP2A in liver cancer tissues and normal paracancer tissues was analyzed by GEPIA database.HepG2 and Huh7 cell lines with TOP2A knockdown were constructed by siRNA technology,and the lentiviral vector overexpressing TOP2A was transferred into Huh7 cells to construct TOP2A overexpressed cell lines.CCK8 assay was used to investigate the effects of TOP2A knockdown and overexpression on the proliferation of HCC cells.The effect of TOP2A on migration of HCC cells was observed by scratch healing experiment.Annexin-FITC and PI double hair dyeing were used to observe the effect of TOP2A on hepatoma cell apoptosis.Annexin V-Alexa Fluor 647 and PI double staining were used to detect the effect of TOP2A on hepatocellular carcinoma cell cycle.RNA transcriptome sequencing technology was used to detect the change level of Huh7 gene in HCC cells after TOP2A knockdown,and the differentially expressed genes were screened out for GO functional enrichment analysis and KEGG pathway enrichment analysis.HepG2 and Huh7 cells were treated with acetylase inhibitor C646,HepG2 and Huh7 cells were treated with deacetylase inhibitor TSA,and the relationship between the expression levels of H3K9ac and H3K27ac and TOP2A was analyzed.Results:Compared with normal hepatic epithelial cells,TOP2A was highly expressed in HCC cells.Compared with normal adjacent tissues,TOP2A was highly expressed in HCC tissues.Knockdown TOP2A inhibited the proliferation of HepG2 and Huh7 hepatoma cells,blocked the cells in G2/M phase,inhibited the migration of cells,and enhanced the apoptosis ability.Overexpression of TOP2A can promote the proliferation,cycle and migration of Huh7 HCC cells,and reduce the apoptotic ability.With Fold≥0.5 and P ≤0.01 as criteria,191 genes were significantly up-regulated and 413 genes were significantly down-regulated,and the differential genes were significantly enriched in the P53 pathway.Rt-qpcr results showed that P53 was significantly up-regulated and CDK1 was significantly down-regulated after TOP2A knockdown.Western Blot results showed that the levels of H3K9ac and H3K27ac increased after TSA treatment of HepG2 and Huh7 cells,and the expression of TOP2A also increased.After C646 treated HepG2 and Huh7 cells,the level of H3K9ac decreased,and the expression of TOP2A also decreased.Conclusion:The expression of TOP2A is up-regulated in hepatocellular carcinoma cells,and knocking down TOP2A inhibits the proliferation,migration and apoptosis of HepG2 and Huh7 cells.Overexpression of TOP2A promoted Huh7 cell proliferation and inhibited apoptosis.Knocking down TOP2A may lead to G2/M phase arrest of Huh7 cells and inhibit cell proliferation by affecting P53 pathway.The high expression of TOP2A in HCC cells may be related to H3K9ac enrichment.