The Influence Of Emodin To Hepatocellular Carcinoma Cell HepG2 Proliferation And Apoptosis And The Tumor Suppressor RASSF10

Objective:Effects of emodin on proliferation and apoptosis of human hepatocellular carcinoma cell line HepG2 and the expression of tumor suppressor RASSF10,and to explore its action mechanism.Methods:The hepatocellular carcinoma cells HepG2 were cultured in vitro and randomly divided into five groups,respectively,to be treated with 0、20、40、80umol/L emodin and 5umol/L 5Aza-cdR,It is negative control group,low dose group,medium dose group,high dose group and positive control group,Collecting HepG2 cells of five groups after 0h,24 h,48 h,72 h and detecting the cells proliferation inhibition rate by MTT detection.Collecting HepG2 cells of five group after 48 hours,the apoptosis rate was detected by Flow Cytometry;The methylated specific PCR(MSP)was used to detect the proliferation of RASSF10 gene promoter methylation and non-methylation.The expression of RASSF10,DNMT1,DNMT3 a and DNMT3 b were detected by Western blot(PCR).Results: 1.According to the MTT result,compared with the blank control group,the HepG2 cells OD value have obvious decreased after the drug effects 24 h,48 h and 72 h;while the other group compared with the blank control group(P ≥0.05),The OD value at the same time points in the experimental group cells were lower than the negative control group(P<0.05),With the increase of drug concentration and time,the difference between groups was statistically significant(P<0.05).2.According to the Flow cytometry(FCM)result,the each group cell apoptosis rate is respectively:(6.77±0.21)%、(10.08±0.56)%、(14.87±0.78)%、(27.9±0.29)%、(21.05±0.35)%,compared with the blank control group,the flow cytometry results showed that the apoptosis rate of HepG2 cells in emodin group was increased(P <0.05),and the apoptosis rate increased obviously with the increase of drug concentration,the contrast between groups was statistically significant.3.According to the MSP results,the methylated PCR in the negative control group was positive,and non-methylated PCR amplification was negative.The methylated PCR and non-methylated PCR amplification are positve in the low dose group and medium dose group.While the methylated PCR in the high dose group and positive control group was negative,and non-methylated PCR amplification was positive.4.Western blot results showed that compared with the blank group,the level of RASSF10 protein in HepG2 cells of emodin group increased,while the Level of DNMT1 and DNMT3 a protein decreased in a dose-dependent manner(P <0.05),but the level of DNMT3 b protein remained unchanged(P <0.05).Conclusion:1.Emodin can inhibit hepatocellular carcinoma cells HepG2 proliferation and promote its apoptosis.2.Emodin keep the demethylation of RASSF10 gene in hepatocellular carcinoma cell line HepG2 by inhibiting the expression of DNMT1 and DNMT3 a.