Study On The Effect Of RASSF1A Methylation On Hepatocellular Carcinoma

Objective To investigate the expression of RASSF1 A gene methylation in human hepatoma carcinoma cell(HCC)and to explore the effect of DNA methylation inhibitor5-Aza-2,-deoxycytidine(5-Aza-Cd R)on the proliferation of human hepatoma carcinoma cell line Hep G2 cells and the expression of tumor suopressor gene RASSF1 A m RNA.Methods Hepatoma cell lines and normal liver cell lines cultured in vitro,MSP(methylation-specific)method was used to detect the methylation level of RASSF1 A gene in HCC cell lines Hep G2,Hep3 B,sk-hep1 and Normal liver cell line LO2,respectively.HCC cell lines Hep G2 as the research object was treated with different concentrations 5-Aza-Cd R(0.5umol/L,5umol/L,50umol/L)as the control group was treated with 5-Aza-Cd R(0umol/L).The CCK-8 method was used to detect the changes of cell proliferation in two groups experimental group to detect the effect of 5-Aza-Cd R on expression m RNA level of RASSF1 A gene by RT-PCR before and after 5-Aza-Cd R treatment.Results Human hepatocellular carcinoma cell line HepG2,Hep3 B,sk-hep1 were detected by methylation of RASSF1 A gene,the methylation of RASSF1 A gene was not detected in normal liver cell line LO2,suggesting that the carcinogenesis of hepatocellular carcinoma is associated with the methylation of RASSF1 A gene.5-Aza-Cd R can inhibit the growth of liver cancer cell line Hep G2.After 72 hours of treatment,the inhibition rate was the most obvious,and with the increase of drug concentration,the inhibition rate of proliferation was more obvious,which indicated that demethylation could inhibit the growth of cells(F=44.164,P < 0.01),showing that demethylation could inhibit the growth and proliferation of human hepatoma cell line Hep G2.After 5-Aza-Cd R treatment,the expression of RASSF1 A in the experimental group was significantly increased compared with the control group,which was dose dependent in m RNA.At 50 umol/L,the demethylation effect was the most obvious.50 umol/L 5-Aza-Cd R,48 h,72h 24 h RASSF1A m RNA gene expression levels were(0.2798±0.0048?0.3678±0.0161?0.3873±0.00105),there was statistically significant difference compared with control group(P < 0.05),indicating that the methylation of RASSF1 A gene in Hep G2 cells can increase the expression of m RNA.Conclusions 1.HCC is associated with the methylation of a novel tumor suppressor gene RASSF1 A.2.5-Aza-Cd R can inhibit the growth and proliferation of Hep G2.cells in adose-dependent manner,and its mechanism may be related to the regulation of RASSF1 A gene promoter methylation,which may be related to the upregulation of RASSF1 A gene expression in m RNA.