| BackgroundHepatocellular carcinoma is one of the common malignant tumors in our country and one of the main types of primary hepatocarcinoma. However, its pathogenesis has not been completely elucidated, and progression of hepatocellular carcinoma is a process that involves multiple genetic changes, multi-factor, multi-step process. Recent studies have found that epigenetic alterations in hepatocellular carcinogenesis, development process plays an important role. Epigenetic phenomena was known including DNA methylation, genomic imprinting, maternal effects, gene silencing in nucleolar dominance, dormant transposons activation and RNA edit. The tumor suppressor gene RUNX3promoter region of the CpG island is methylated, leading to the gene silencing, which become a research focus of hepatocellular carcinoma related to the occurrence and development mechanism and treatment of in recent years.Research reported that RUNX3gene in hepatoarcinoma cell is obviously down-regulated in the mRNA level and protein level because of CpG Island promoter hypermethylation.5-Aza-2’-deoxycytidine(5-Aza-dC) is a nucleotide such methytransferase inhibitors,which is also a DNA methyltransferase specific inhibition, and it can lead to DNA methylation degree drop, then reversed the silencing genes to restart to express. Related studies in RUNX3gene CpG island methylation in hepatocarcinoma cell have been reported, while the study on5-Aza-dCacting upon the gene in methylation status of hepatocarcinoma cell line SMMC-7721is seldom.ObjectiveTo explore the effect of demethylation agents5-Aza-dC on the expression and the promoter region methylation status of the RUNX3gene, cell growth cycle and drug sensitivity in SMMC-7721cells.Materials and methodsSMMC-7721cells were given by the Central Laboratory of Henan Tumor Hospital and cultured in the RPMI-1640medium (10%fetal bovine serum,100U/ml penicillin,100mg/ml). Culture fluid was placed at37degrees, saturated humidity cell culture box.. Every2to3days for the fluid passage1times. And cells were divided into control group (no treatment with5-Aza-dC) and5-Aza-2’-deoxycytidine treatment group.1The effects of different concentration of5-Aza-2’-deoxycytidine on RUNX3gene the promoter region methylation status in human hepatocarcinoma cell SMMC-7721:Hepatocarcinoma cells SMMC-7721were cultured in respectively0.5μmol/L、1.5μmol/L、4.5μmol/L、13.5μmol/L5-Aza-2’-deoxycytidine culture liquid for72h and DNA was extracted.Then according to methylation specific PCR technology, methylated and non-methylated detection of primers were designed respectively, and methylation specific PCR (MSP) technique was performed to invest the effect of5-Aza-dCon methylation status of RUNX3gene promoter region in two groups of hepatocellular carcinoma cells.2The effects of different concentration of5-Aza-2’-deoxycytidine on RUNX3gene mRNA level in human hepatocarcinoma cell SMMC-7721:Hepatocarcinoma cells SMMC-7721were cultured in respectively0.5μmol/L、1.5μmol/L、4.5μmol/L、13.5μmol/L5-Aza-2’-deoxycytidine culture liquid for72h and RNA was extracted, RT-PCR(Real-time PCR) technique was used to detect the effect on expression of RUNX3mRNA in two groups of hepatocellular carcinoma cells..3The effects of different concentration of5-Aza-2’-deoxycytidine on cell cycle in human hepatocarcinoma cell SMMC-7721:Hepatocarcinoma cells SMMC-7721were cultured in respectively0.5μmol/L、1.5μmol/L、4.5μmol/L、13.5μmol/L5-Aza-2’-deoxycytidine culture liquid for72h and collect cells, the flow cytometry (FCM) technique was used to invested the effect on cell cycle in two groups of SMMC-7721hepatoma cells.4Data analysis:statistical software SPSS18.0was used for data analysis, data was showed as mean±Standard deviation(-x±s), p<0.05was considered that the difference was statistically significant.Results1The effects of different concentration of5-Aza-2’-deoxycytidine on RUNX3gene the promoter region methylation status in human hepatocarcinoma cells SMMC-7721:MSP results showed that RUNX3gene promoter region was at high methylation status in the control group of hepatocarcinoma cells SMMC-7721, and RUNX3gene promoter region methylation status was increasingly reversed with the concentration of5-Aza-dC gorwing,after hepatocellular carcinoma cells SMMC-7721were cultured in respectively0.5μmol/L.,1.5μmol/L、.4.5μmol/L、13.5μmol/L5-Aza-2’-deoxycytidine culture liquid for72h.2The effects of different concentration of5-Aza-2’-deoxycytidine on RUNX3gene the promoter region methylation status in human hepatocarcinoma cells SMMC-7721:Real-time PCR(RT-PCR) detection results showed that RUNX3mRNA level was not detected in control group of hepatocarcinoma cells SMMC-7721, after hepatocellular carcinoma cells SMMC-7721were cultured in respectively0.5μmol/L、1.5μmol/L、4.5μmol/L、13.5μmol/L5-Aza-2’-deoxycytidine culture liquid for72h.,RUNX3mRNA expression levels were0.33±0.04,0.94±0.08,1.23±0.05and1.90±0.03,RUNX3mRNA levels were increased in the control group.And results reflected a growth with concentration dependent manner.RUNX3mRNA level and 5-Aza-dC concentrations were positively correlated (r>0.95, P<0.05), the differences were statistically significant.3The effects of different concentration of5-Aza-2’-deoxycytidine on cell cycle in human hepatocarcinoma cell SMMC-7721:FCM results showed that after treatment of different concentrations of5-Aza-dC for72h,SMMC-7721hepatocarcinoma cell ratio in S phase increased with the drug concentration suggesting that5-Aza-Dc can induce SMMC-7721hepatocarcinoma cells arrested in S phase; S phase cell ratios increased in a concentration dependent manner. There was a a significant positive correlation between S phase cell ratio and the concentration of5-Aza-dC(r>0.95, P<0.05).ConclusionRUNX3gene promoter region was at high methyl ation status in hepatocarcinoma cells SMMC-7721.5-Aza-dC is able to reverse RUNX3gene promoter region methylation status in hepatic cancer cell SMMC-7721, upregulate RUNX3gene expression,and induce hepatoma cells to be arrested in S phase.And there was a significant positive correlation between the effect and the drug concentration. |
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