Prognostic Significance Of RASSF1A, APC, WIF-1 Methylation In Paraffin Embedded Tissues Of HCC

Objective: Hepatocellular carcinoma is one of the most common malignancy tumor with rising incidence, while carcinogenesis progress remains unclear. The aberrant promoter methylation of the suppressor genes are associated with tumor development. The tumor suppressor genes of RASSF1A and APC are belonged to the Ras and Wnt signaling pathway which has a role in HCC carcinogenesis. Suppressor gene WIF-1 is also belonged to the Wnt signaling pathway with few research papers. Fresh tissues were adopted widely in DNA methylation research for RASSF1A, APC, WIF-1, but fresh tissues had a promblem for conservation. In this study, we assess the promoter methylation degree of RASSF1A, APC, WIF-1 genes in paraffin embedded HCC tissues by using methylation specific PCR, and the association of the clinical characteristics with methylation degree were analyzed. Our results suggested that RASSF1A, APC and WIF-1 genes involved in HCC process.Methods:1 Patients: Ninety-seven cancerous tissues and twenty-six adjacent non- cancerous cirrhosis tissues of HCC patients were collected from the Fourth hospital of Hebei Medical University between 2002 and 2010. There were eighty-one males and sixteen females in HCC group, the age from twenty-four to seventy-five years old, the average age was 55.69±10.21. the diagnosis were confirmed by two pathologist without clinical information of patients. Then the case history, laboratory tests, clinical pathology data, family history were recorded, and followed up the survival status.2 Methylation specific PCR: The total DNA from the paraffin-embedded tissues was extracted with phenol/chloroform method, and modified with sulfite methods and purified with Wizard DNA kit, methylation specific PCR of these three suppressor genes was run, the methylation status was assessed subsequently.3 Statistics analysis: All of the datas were analysised by SPSS13.0 software, difference between groups were compared with Chi-square test or Fisher's Exact Test, Kaplan-Meier method was used to evaluate survival time. The prognostic factors were analyzed by log-rank analysis. Two-sided tests were used to determine significance, and P<0.05 was considered as statistical significantance.Results:1 Comparing the methylation of suppressor genes RASSF1A, APC, WIF-1 in cancerous and adjacent non-cancerous cirrhosis tissues. Positive ratio of RASSF1A gene methylation in cancerous tissues and adjacent non-cancerous cirrhosis tissues were 65 of 97(67.01%) versus 11 of 26(42.31%), significant difference between two groups was exist. Positive ratio of APC gene methylation in cancerous tissues and adjacent non-cancerous cirrhosis tissues were 62 of 97(63.92%) versus 11 of 26(42.31%), significant difference between two groups was exist. Positive ratio of WIF-1 gene methylation in cancerous tissues and adjacent non-cancerous cirrhosis tissues were 45 of 97(46.39%) versus 2 of 26(7.69%), significant difference between two groups was exist.2 Comparing RASSF1A, APC, WIF-1 gene's methylation status and the clinical character in cancerous tissues. No difference for RASSF1A methylation frequency exist between the male and female patients. While statistical difference for APC and WIF-1 appeared between different sex with 37.5% versus 69.14% and 18.75% versus 51.85%, respectively. Significant difference for the suppressor gene RASSF1A in HBsAg positive and negative groups were confirmed as well as in cirrhosis and non-cirrhosis groups, but not for APC and WIF-1 genes.3 Association of RASSF1A, APC, WIF-1 gene's methylation status and the pathological character in cancerous tissues. Significant difference for suppressor gene WIF-1 in tumor capsular positive and negative groups, but not for RASSF1A, APC genes. The frequency of the methylation were not correlated with tumors'number, size, portal vein tumor embolus and clinical stages.4 The internal relationships among RASSF1A, APC and WIF-1 gene's methylation.In cancerous tissues, the frequency for non-methylation of all the three suppressor genes, two genes methylation and one gene methylation was 11.34%, 64.95 and 88.66%, respectively. No internal relationships could be found among these three genes with Pearson's test.5 Association of the methylation status for suppressor genes RASSF1A,APC,WIF-1 with the survival time.Followed-up was ended in March 2011, the survival period from 1 to 105 months, 9 of 97 cases of HCC patients were lost to follow up. The survival rate for one, two and three years were 57.3%, 42.1% and 35.4%, respectively. No significant difference between methylation positive and negative groups for these three genes referring to survival time could be found.Conclusions:1 The methylation of the suppressor genes RASSF1A, APC, WIF-1 in HCC paraffin embedded tissues are detected, the method is reliable and feasible.2 The aberrant promoter methylation ratio of the three suppressor genes (RASSF1A, APC, WIF-1) are significant difference in cancerous tissues and adjacent non-cancerous cirrhosis tissues, which suggests that the methylation of the suppressor genes RASSF1A, APC, WIF-1 may play an important role in HCC carcinogenesis.3 Significant difference for the suppressor gene RASSF1A in HBsAg positive and negative groups are confirmed as well as in cirrhosis and non-cirrhosis groups, but not for APC and WIF-1 genes. Demonstrating in some degree that the methylation of suppressor gene RASSF1A can be used to screening early HCC (patients with hepatitis infection or cirrhosis).4 There is no statistics significance between the methylation status of the suppressor genes RASSF1A, APC and WIF-1 and the survival ratio in HCC tissues, suggesting that the methylation of the three suppressor genes may not be used as the prognostic factor for HCC.