The DNA Methyltransferase 3B Silenced To Increase XAF1 Expression Promote Apoptosis Of Hepatocellular Carcinoma Cells HepG2

Objective To explore the X chromosome-linked inhibitor of apoptosis-associated factor 1(XAF1) expression after DNA methyltransferase 3B silencing and its mechanism of Hep G2 cells apoptosis.Methods The hepatocellular carcinoma cells Hep G2 were divided into the experimental group and the negative control group, blank control group. Experimental cells were transfected the interference plasmid of DNA methyltransferase 3B(DNMT3B-si RNA), the negative control group cells were transfected empty plasmid, blank control group cells without treatment. We transfected the DNMT3B-si RNA to human hepatocellular carcinoma Hep G2 cells by liposome transfect methods.To observe the transfection rate under the fluorescence microscope.We selected the cells of the largest transfection rate timing for subsequent experiments, Methylation specific PCR(MSP) analysis of XAF1 gene promoter methylation; To use Western blot detect the expression of DNMT3 B, XAF1, Caspase3 protein; after 24 h,48 h,72 h,96 h we collected the Hep G2 cells of three groups, and detected the cells proliferation inhibition rate by MTT detection; Flow Cytometry detected the cell apoptosis rate after the DNMT3B-si RNA transfection.Results In this experiment, gene sequencing results showed that the recombinant plasmid were constructed successfully.To observe Hep G2 cells transiently transfect efficiency, it is about 70%-80%. According to the MSP results,the XAF1 gene promoter methylation was existent, compared with negative control group, Hep G2 cells XAF1 gene promoter methylation level decreased significantly after DNMT3 B silenced, the demethylation expression were enhanced(all P<0.05).Western blot showed that compared with negative control group, after the DNMT3 B silenced,the expression of DNMT3 B in experimental group hepatocellular carcinoma cells were down-regulated significantly, and XAF1 protein expression level were upregulated, Caspase3 expression were increased(all P<0.05), while the negative control group compared with blank control group,the difference of these indicates were no statistically signification(P?0.05). According to the MTT result, compared with negative control group, the Hep G2 cellular proliferation inhibition rate have obvious enhanced after the DNMT3 B silenced 24 h(7.82±3.01%)?48 h(16.18±4.22%)?72 h(25.66±3.20%)?96 h(22.8±2.69%). And the negative control group compared with blank control group, Hep G2 cells proliferation inhibition rate at each time point were no signification(P?0.05). Flow cytometry results showed that compared with negative control group(4.00±0.76%), after DNMT3 B silenced, the Hep G2 cells apoptosis rate(15.30±0.67%)increased significantly(P<0.05).Conclusion Silence the expression of DNMT3 B can inhibit hepatocellular carcinoma cells Hep G2 proliferation and promote its apoptosis, this process is closely related to the XAF1 upregulation after its demethylation.