The Studies On The Mechanism Of Epigenetic Changes And Regulative Expression Of Genes In Human Hepatocarcinoma

Objective:In order to explore the molecular mechanism of different gene regulation in hepatocarcinogenesis,we study the microsatellite instability(MSI),loss of heterozygosity(LOH) and methylation of p57^kip2 gene in human hepatocellular carcinoma(HCC) by using genetic diversity analysis and methylation-specific PCR (MSP) methods.Meanwhile,the relationship and potential regulative mechanism were studied between CDK6 and XIAP expression in mRNA and protein levels of hepatocacinoma in vitro.Methods:1.Studies on genetic instability of p57^kip2 gene in human HCC:The expression of P57^kip2 protein and mRNA were detected by using immunohistochemistry staining and in situ hybridization(ISH) in 30 cases of pericancerous cirrhosis and HCC.LOH and MSI of ten microsatellite loci(D11S1397,D11S1318,D11S4046,D11S922,THO1, D11S2359,D11S1760,D11S1338,D11S569 and D11S1397) were detected where the p57^kip2 gene is located in the area of 11p15.5 to 11p15.1 by using PCR-Polyacrylamide gel electrophoresis-silver staining method in 30 cases of HCC. Then,the PCR products were genotyped to confirm the results of LOH and MSI.The relationship between genetic instability and the expression of P57^kip2 protein and mRNA were analysis.2.Studies on methylation of p57^kip2 in human HCC:The CpG island methylation of p57^kip2 gene in promotor regions were detected in 30 cases of pericancerous cirrhosis and HCC by using methylation-specific PCR(MSP).The MSP products were detected again for DNA sequence analysis to confirm the results of methylation. And the relationship between the methylation of p57^kip2 and the expression of their protein and mRNA were also analysis.3.The regulative mechanism between XIAP and CDK6 expression in HepG2 cell line:The expression of CDK6 and XIAP mRNA and protein were detected by using RT-PCR and Real-time PT PCR and Western Blots method,after separate knockdown CDK6 and XIAP with small-interfering(si) RNAs in HepG2 cell line in vitro.The relationship and potential regulation between CDK6 and XIAP pathway were analysis.Results:1.Studies on genetic instability of p57^kip2 gene in human HCC: Immunohistochemistry staining showed that there were no expressions of P57^kip2 proteins in normal liver tissue.Expressions of the protein in precancerous cirrhosis and hepatocellular carcinoma were 56.67%(17/30) and 63.33%(19/30) respectively. The positive rates in high,moderately and poor differentiated HCC were 0%(0/1), 54.1%(13/24) and 80%(4/5).There was significant difference between the expressions of P57^kip2 proteins and the degree of differentiation of HCC.ISH study represented that there were no expressions of p57^kip2mRNA in normal liver tissue. Expressions of mRNA both in pericancerous cirrhosis and hepatocellular carcinoma were 26.67%(8/30).The positive rate in high,moderately and poor differentiated HCC were 0%(0/1),20.8%(5/24) and 60%(3/5).There was also significant difference among these three groups of HCC tissues.There was positive correlation between expression of p57^kip2 mRNA and expression of P57^kip2 protein.LOH and MSI detected showed that LOH was identified only by TH01(7/30,23.3%),D11S2359(5/30,16.6%) and D11S569(1/30,3.3%) in 30 cases on ten microsatellite loci,and there were 4 microsatellite loci including D11S1760(3/30,10%),D11S922 (2/30,6.7%),D11S2351(4/30,13.3%) and D11S569(1/30,3.3%) showing MSI.The loss of expressions of p57^kip2mRNA and protein mainly related to the LOH of TH01 and MSI of D11SD11 and S1760.2.Studies on methylation of p57^kip2 in human HCC:MSP detected showed that there were 6 hypermethylation in 30 cases of HCC,the methylation rate was 20% (6/30).There was no hypermethylation in pericancerous of HCC.There was negative correlation between the loss of p57^kip2 expression and the methylation of their promotor region.3.The regulative mechanism between XIAP and CDK6 expression in HepG2 cell line:XIAP knockdown of HepG2 cell line by using XIAP siRNA transfected also can obviously reduce the CDK6 mRNA expressions.But the expression of XIAP mRNA did not change in CDK6 siRNA transfection group.CDK6 protein reduced prominently after siRNA transfection,and re-expression can be seen after MG132 treated.But XIAP expression wasn't affected by CDK6 siRNA.Conclusions:1.The disorder expressions of p57^kip2 mRNA and protein indicated that p57^kip2 may be involved in hepatocarcinogenesis and prognosis.However,it need further studies wether p57^kip2 play as a role of tumour suppressor gene in HCC.1.1 The ten microsatellite loci where the p57^kip2 gene is located in were not the frequent loci for LOH and MSI,but the loss of expressions of p57^kip2mRNA and protein mainly related to the LOH of TH01 and MSI of D11SD11 and S1760.1.2 The hypermethylation of p57^kip2 might not lead to the loss of expression of p57^kip2 mRNA and protein.2.XIAP siRNA can obviously reduce the expression of CDK6 mRNA.It is first time to confirm there is regulative relationship between XIAP and CDK6.But CDK6 siRNA don't interfere the expression of XIAP both in mRNA and protein level.The detail mechanism needs further studies.