Research On The Status Of HMLH1 Promoter Methylation In Peripheral Blood Of Rheumatoid Arthritis Patients And The Significance Of DNA Methylation In Forensic Medicine

Objective: Rheumatoid Arthritis(RA) is a kind of chronic and systematical deseases with the appearance of symmetrical and mutiple arthritis,it is ordinary in china with an incidence rate of 0.3%.Till now,etiopathogenisis of rheu- matoid arthritis hasn't been illuminated.Recently,some researches indicated that low DNA methylation was frequent- tly found in rheumatoid arthritis patients'genome,which might lead to the absence of some genes'expression and play a role in the development of the desease.DNA methylation is one of the most intriguing chemical phenomena affecting the genome. It is essential in prokaryot- es,dispensable in lower eukaryotes such as Saccharomyces c- erevisiae, yet present and presumably important in mammals. As gene expression is abnormal in cancer and DNA methyla- tion plays a role in controlling gene expression, it was supp- osed that aberrant DNA methylation might be responsible for expression differences.DNA mismatch repair(MMR) is an important system in avoidance from mutation,which maintains the stability of genome through correcting base mispairing in normal DNA metabolism and regulating apoptosis coming after DNA impairment.MMR is essential for duplicating and prolifer- ating exactly.if MMR is abnormal,the frequency of cell mutation may increase more than 100 times. DNA mismatch repair in human cells depends on the participati- on of some productions of human MMR genes,therefore, mutation in any kind of MMR genes that lead to the function deprivation of its production may result in the abnormality, discrepancy and deprivation of DNA mismatc- h repair function.Up to now,there are 9 mismatch repair genes: hMLH1, hMLH3,hPMS1,hPMS2,hMSH2,hMSH3,hMSH4,hMSH5 and hMSH6.Among them, hMLH1 is one of the most important mismatch repair genes,its principal role is keeping the integrity and stability of genetic substance and true of duplication.If this gene was defective which result in MSI and probably led to the instability of genome and happeness of desease.The absence of hMLH1 expression is concerned with promoter methylation.Transcriptional silencing of proapop- totic genes,including the DNA mismatch repair (MMR) gene hMLH1,due to aberrant CpG island methylation has been implicated in acquired resistance to chemotherapeutic drugs in vitro. hMLH1 promoter methylation is early molecular activity of deactivation,which can decrease the mRNA and protein expression,affect its normal renovation function,make the relative gene mutation can not be corrected in time,at last,lead to the development of desease.However, what's the methylation status of hMLH1 promoter in RA patients?there is no report at home and abroad. In order to study on this problem,we use human peripheral blood (sodium citrate anticoagulation) as research subjects to observe the status of hMLH1 methylation,mRNA and protein expression. Recently, DNA methylation is hot in the research of forensic medicine. according to the character of DNA methylation,its application in forensic medicine is significant.Methods: Adopting peripheral blood of RA patients and healthy persons, diverging mononuclearcells to exrtact RNA;whole blood to exrtact DNA and nucleoprotein protein.Using RT-PCR and Western Blot methods to detect hMLH1 mRNA and protein expression in human peripheral blood respectively.Using the Gel-pro analysis software to carry through semi quantitative analysis.Adopting Sodium Bisulfite method to deal with DNA, Methylation-specific PCR(MSP) method to detect the status of hMLH1 promoter methylation.the experiment is falled into two groups:control group and experimental group.Using SPSS11.5 statistic analysis software to processχ2 or Fisher's test and Spearman correlation analysis,Using Wilcoxon W test to compare means.Results: 1 hMLH1 mRNA were expressed in human peripheral blood of all 38 heathy persons,17 hMLH1 mRNA were not expressed in 33 RA patients.hMLH1 protein were expressed in human peripheral blood of all 38 heathy persons.while,22 hMLH1 protein were not expressed in peripheral blood of 33 RA patients.and hMLH1 protein expression was agree with its mRNA expression. Using Wilcoxon W test,The positive rate of hMLH1 mRNA and protein in RA patients was significantly lower than that in healthy persons(P<0.05).2 The positive rate of hMLH1 promoter methylation in RA patients and healthy persons were 90.9%(30/33)and 13.2%(5/38)respectively, there was significantly different between the two groups(χ2=42.717,P<0.05).3 Prmoter methylation and mRNA,protein expression of hMLH1 gene showed significantly negative correlation (r=-0.866, P<0.01)(r=-0.541, P<0.01).4 There was not enough evidence to say that hMLH1 promoter methylation,mRNA and protein expression were correlated with different age,sex,ESR,CRP and RF of RA patients(P﹥0.05).5 The application of DNA methylation in forensic medicine is significant.Conclusions: hMLH1 promoter is hypermethlated in peripheral blood of RA patients. The hypermethylation affect the mRNA and protein expression of hMLH1,Which might take a role in the development of RA .The application of DNA methylation in forensic medicine is significant.