The Experimental Research On DNA Methylation Profile In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world and among the most fatal of human neoplasms, but the molecular mechanisms of hepatocarcinogenesis are primarily unknown. Aberrant promoter hypermethylation has been shown to be a common event in HCC mainly due to functional loss of the tumor suppressor genes(TSGs). In this study, we established the DNA methylation profile of HCC in an effort to explore the clinical implication of DNA methylation profile and molecular mechanism of HCC. The samples of 60 paired HCC and corresponding tissue adjacent to tumor, 16 cases of liver cirrhosis(LC), 5 cases of chronic hepatitis(CH) and 5 cases of normal liver(NL) were collected in Tianjin and 8 tumor suppressor genes frequently silenced in digestive tumor by promoter hypermethylation were selected. The promoter methylation status of the 8 genes was investigated using methylation-specific polymerase chain reaction for all samples. The clinicopathological data of HCC were also obtained in order to evaluate the implication of HCC methylation profile. Our result showed that methylation of the 8 tumor suppressor genes was a quite frequent event in HCC. The methylation profile based on our HCC samples was charactered with Chinese region and HBV-associated pathogeny. Methylation of RASSF1A(95%),APC(90%),GSTP1(73.3%),p16(65%),RIZ1(61.6%) and MGMT(60%) was more frequent in HCC than in tissue adjacent to tumor(p<0.05), which indicated tumor-specificity of the 6 genes methylation. Methylation of MGMT(41.6%),GSTP1(40%) and RIZ1(25%) was significantly more frequent in tissue adjacent to tumor than in liver cirrhosis(p<0.05). Different methylation frequency in HCC, tissue adjacent to tumor and liver cirrhosis showed gradually epigenetic alteration in hepatocarcinogenesis. Furthermore, methylation status of MGMT, GSTP1 and RIZ.1 would be very promising predict factor for HCC risk assessment and early diagnosis. Further results showed that p16 methylation was more frequently observed in HCC from older patient, which indicates the dual effect of p16 methylation in age and tumorigenesis. The frequency of MGMT methylaiton tended to be higher in massive HCC than in other gross type. Patients with MGMT methylation in tumor had a shorter disease free survival. These results showed that MGMT methylation might also serve as a potential biomarker for prognostication of HCC. Field cancerization is an animate theory about tumorigenesis. The theory not only can be used to explain how multiple tumors develop but also has important clinical implication in risk assessment, early detection, molecular margin and chemoprevention. Until now, field cancerization has been described in almost all organ systems, but has not been thoroughly studied in the liver. In analysis of methylation profile in HCC,tissue adjacent to tumor and cirrhosis, the fact that epigenetical alternations correlated with HCC but not with LC or CH exist in non-cancerous adjacent tissue strongly suggested the presence of field cancerization in liver. Furthermore, we demonstrated that the methylation status of HCC and adjacent tissue were accordant in 39(65%) of the cases, but inconsistent for 1-3 genes in 21(35%) of cases, which showed complicated clone origin in HCC. In addition, considering corresponding shorter disease free survival, RIZ1 methylation in adjacent tissue would have the implication of prognostic assessment. We also found that local recurrence but not margin recurrence was very common in patients with RIZ1 methylation in adjacent tissue and small HCC, which indicated that second field tumor based on field cancerization played an important role in HCC recurrence.Different from mutation and deletion, DNA hypermethylation don't change DNA sequence and thereby can be reversible. Demethylation and consequent re-activation of TSG is an attractive avenue for the development of novel therapeutics. 2'deoxy-5-azacytidine(DAC) has long been used experimentally to induce cell differentiation by demethylating global genome without specificity. However, due to its specific pharmacokinetic feature and the potential probability leading to tumor, its clinical use is just limited in some haematopoietic tumor. More effort is still needed for this kind of drug to explore the mechanism, optimize clinical protocol and extend the scope of utility. In addition, many studies have also been looking for some other hypomethylation agents with high specificity and low toxicity. Matrine, as a traditional Chinese medicine, has been proved to have many mechanisms to interrupt tumor pathway such as inducing tumor differentiation and enhancing expression of TSG. But whether matrine can be used as a hypomethylation agent is not clear. To study the tumor-induced mechanism of DAC and matrine, we treated HepG2 cell with DAC and matrine in different concertration and detected the change of HepG2 in cell survival ratio, morphology, DNA methylation, gene transcription and protein expression. Our results showed that DAC could promote benign differentiation, reactivate numerous methylation-selienced genes and arrest cell cycle in G2/M phase. But its toxicity was also obvious and much time was needed for it to show the efficacy. Matrine had two different antitumor mechanisms, tumor-induced in low concentration and toxicity in high concentration. Matrine in low concentration could arrest cell cycle in G0/G1 phase. However, it could not reverse the hypermethylation of silenced TSG, which showed that demethylation wasn't included in the tumor-induced mechanism of matrine. We also found that matrine in low concentration could upgrade the expression of pl6 and p53 in concentration dependence. Combined effective pathway of the two gene and G0/G1 arrestment, we predicted that enhancing expression of p16 and p53 induced by matrine might play an important role in its antitumor mechanism.