| Objective: The incidence of hepatocellular carcinoma (HCC) isincreasing, with the poor prognosis, however the pathogenic mechanismm isnot entirely clear. With the development of molecular biology and detectivetechnology, more and more attentions focused on the role of the methylationof tumor suppressor genes in tumorigeness and development. The RINGfinger proteins with the ubiquitin ligase activity play a role in cell growth anddevelopment by regulating the ubitination of the target protein substrate.RNF180is a kind of menbrane conjunction E3ligase which includes RINGfinger domain and abundant of Cysteine/Histidine (C3HC4). RNF180gene isa newly discovered tumor suppressor gene. According to the report, promoterhypermethylation of the RNF180gene in gastric cancer has an important rolein the genesis and development, which will be the potential clinical andbiological indicators of gastric cancer patients. Although the relationship ofmethylation of RNF180and gastric cancer is clear, there are few reports of therelationship with other tumors. Accordong to the above data we want to studythe relationship of the RNF180gene methylation and hepatocellularcarcinoma. So we assess the promoter methylation degree and mRNAexpression of RNF180gene in fresh HCC tissues by using methylationspecific polymerase chain reaction (Methylation specific PCR, MSP) andRT-PCR, and analysis for the association of the clinical character, pathologydata and prognosis of survival. According our results we could offerfoundation to clinical application of methylation detective of RNF180gene,interpret the effect about development of RNF180gene's DNA, consummatethe anti-oncogene spectrum of methylation modification, and provide theexperiment evidence of the RING-finger family protein ubiquitin E3enzyme's biological function.Methods:1Patients: Tissue samples were collected at the Fourth Hospital of HebeiMedical University (China) from50HCC patients who underwent HCCresection in the Department of Hepatobiliary Surgery beteween2010and2011. There were fouty-five males and five females in HCC group, the agefrom thirty-six to seventy-eight years old, the average age was55.00±8.86.The diagnosis was confirmed by two pathologists without clinical informationof patients. Then the case history, laboratory tests, clinical pathology data,family history were recorded, and followed up the survival status.2Methylation specific PCR: The MSP assay followed the protocol asdescribed previously, with minor change. In brief, sodium busulfite-treatedgenomic DNA was amplified using primers,5'-GACGTGGGGCGAGTAGGGTCGTT-3'(forward)/5'-CCGACCGAAACCAAATCCCTCCGAA-3'(reverse)and5'-GATGTGGGGTGAGTAGGGTTGTT-3'(forward)/5'-CCAACCAAAACCAAATCCCTCCAAA-3'(reverse), forthe methylation and unmethylation of RNF180gene DNA. The genomic DNAfrom the fresh tissues was extracted with phenol/chloroform method, andmodified with sulfite methods and purified with Wizard DNA kit, methylationspecific PCR of the suppressor gene was formed, the methylation status wasassessed subsequently.3Detection of mRNA expression of RNF180gene: The total RNA from thefresh tissues was extracted using RNA extraction Kit, and then revesetranscription, at last to detect mRNA expression.4Statistical analysis: All of the datas were analysised by SPSS13.0software,difference between groups were compared with Chi-square test or Fisher'sExact Test, Kaplan-Meier method was used to evaluate survival time. Theprognostic factors were analyzed by log-rank analysis and COX model.Two-sided tests were used to determine significance and P<0.05wasconsidered as statistical significantance. Results:1Comparing the methylation of tumor suppressor genes RNF180in cancerous,adjacent non-cancerous cirrhosis tissues and the normal liver tissue.Positive ratio of RNF180gene DNA promotor methylation in canceroustissues and adjacent non-cancerous cirrhosis tissues were30of50(60.00%)versus15of50(30.00%), significant difference between two groups was exist.The normal liver tissues do not have methylation.2Comparing the methylation status of RNF180gene's DNA and the clinicalcharacterisis in cancerous tissues.Significant differences of RNF180between the clinical stage. No differencefor RNF180DNA promotor methylation frequency exist referring to the sex,age, HbsAg status, the value of AFP, the fractiongnation of hepatic funcationa.The chistribation of RNA180gene DNA promotor methylation frequency inpatients positive rate referring to gender (P=1.000), age (P=0.470), HBsAgstatus (P=0.724), Child-pugh grading of liver function (P=0.556), the value ofserum AFP (P=0.083), the size of the differences were not statisticallysignificant (P>0.05).3Association of methylation status of RNF180gene's DNA promotor and thepathological characteristic in cancerous tissues.The distribation of the methylation frequency were not association withdifferentiation level, tumors' number, size and portal vein tumor embolus.4Association of mRNA expression status RNF180gene's with the clinicalcharacterisis in cancerous tissues.Expression level of RNF180mRNA in HCC group was sinificantly lowercompared with the cirrhosis tissues. No significantly difference was foundbetween positive and negative groups.Expression level of RNF180gene mRNA in HCC was high expressionreferring to male, younger than60years old, Child A status, single tumor, theless value of AFP, clinical stage I, HbsAg positive.5The corrilation of RN180gene's DNA promotor methylation and theexpression of mRNA.The mRNA expression is lower in positive than nagetive of HCC, and there is the value between them.6Association of the methylation status of RNF180with the survival time.Followed-up was ended March in2012, the survival period from1to23months, none of50cases of the patients was lost to follow up. The survivalrate for six, twelve and eithteen months were86.0%,54.0%and36.0%,respectively. No significant difference exise methylation positive and negativegroups for the gene referring to survival rate could be found. According to theclinicl stage, we found there are sinificantly differences between themethylation positive and the methylation negative in II-III stage.Conclusions:1The positive ratio is higher in hepatocellular carcinoma than that in cirrhosistissues, so we could think the promoter methylation of RNF180may playsome roles during the hepatocellular carcinoma genesis. The methylation ofRNF180DNA promoter may be one of cause for the development of HCC.2The promoter methylation of RNF180gene maybe associate to the clinicalstatus morigenesis, and the RNF180gene promoter meyhylation may reflectpathogenetic condition.3The promotor methylation of the tumor suppressor gene RNF180maybeassociate to prognosis with adwanced stage of hepatocellular carcinoma, andit may be the prognosis and prediction factor of advanced stage ofhepatocellular carcinoma. |
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