The Effect Of5-Aza-CdR On The Proliferation Of Hepatocellular Carcinoma Cells HepG2and The Methylation Of Gene PI3KC3

Objectives:To investigate the effect of demethylating agent5-Aza-2’-deoxycytidine on theproliferation of human hepatocellular cancer cell lines HepG2and themethylationandexpression of human gene PI3KC3. To discuss the primary mechanism ofthe inactivation of human gene PI3KC3in hepatocellular cancer cells, and further explorenew approaches to the treatment of hepatocellular cancer by changing the geneticmethylationof hepatocellular cancer gene PI3KC3.Methods:After two hepatocellular cancer lines HepG2with different degrees of differentiationwere culturedin vitro, the effect of5-Aza-CdR with different concentration(0μmol/L,1μmol/L,5μmol/L,10μmol/L and20μmol/L)on the cell proliferation of hepatocellularcancer lines HepG2was observed at different points of time (at24th hour,48th hour and72th hour) by means of MTT. After the hepatocellular cancer lines HepG2were treatedwith5-Aza-CdRwith different concentration (0μmol/L,1μmol/L,5μmol/L,10μmol/Land20μmol/L) for24hs,48hs and72hs respectively, the expression of the mRNA inPI3KC3was tested with the method of qRT-PCR. the expression of the protein inPI3KC3was tested by means of Western Blotting. After the hepatocellular cancer linesHepG2without drug treatment and those treated with20μmol/L5-Aza-CdR for72hswere collected, two groups of DNA were extracted and the changes in the methylation ofPI3KC3were detected respectively.Results：After the hepatocellular cancer lines HepG2were treated with5-Aza-CdRwithdifferent concentration (0μmol/L,1μmol/L,5μmol/L,10μmol/L and20μmol/L) for24hs,48hs and72hs respectively it was discovered that5-Aza-CdR could inhibit the proliferation of HepG2in vitro.The results showed the dependence in time and dose (P<0.05), andwere statically significant. After the hepatocellular cancer lines HepG2wererespectively treated with the different concentrations of5-Aza-CdR for24hs,48hs and72hs,qRT-PCR method detcted that, PI3KC3mRNA was significantly upregulated by5-Aza-CdR in time and dose-dependent in hepatoma cell lines HepG2(P <0.05), thedifferences were statistically significant; Western blotting method also detected that,PI3KC3mRNA was upregulated by5-Aza-CdR in time and dose-dependent inhepatoma cell lines HepG2(P <0.05), the differences were statistically significant.Results of MSP showed that methylation and unmethylation coexist in the PI3KC3genein the negative control group (without5-Aza-CdR treatment) in HepG2cells, and themethylation level is obviously higher than unmethylation. But the methylation in theexperimental group was significantly decreased after being treated with demethylationdrug5-Aza-CdR(20umol/L) for72hs. The differences were statistically significant.Conclusions: The methylation transferase ihhibitor5-Aza-CdR can inhibit theproliferation of hepatocellular carcinoma cells HepG2, whose mechanism may havesome relation with the expression increase in PI3KC3caused by regulating the geneticdemethylation of the promoter of5-Aza-CdR.