Study Of The Role Of Methylation Of CpG Motifs And Its Regulation Factors In The Pathogenesis Of Systemic Lupus Erythematosus

PartⅠMethylation of CpG motifs and expression of DNA Methyltransferase inthe patients with systemic lupus erythematosus and normal controlsObjective To study methylation of CpG motifs and expression of DNAMethyltransferase(DNMT)mRNA in the patients with systemic lupuserythematosus(SLE)and normal controls,and evaluate their relation.Methods Theperipheral blood lymphocytes were isolated from 38 patients with SLE and 19 normalcontrols,the methylation of CpG motifs was detected by the 5-Methylcytosineantibody and flow cytometry,and the expression of DNMT1,DNMT3A andDNMT3B mRNA was analysed by RT-PCR.Results Methylation of CpG motifs inpatients with active SLE was lower than the control subjects and inactiveSLE(p＜0.01),while there is no statistical difference between and the control subjectsand inactive SLE(p＞0.05),and a negative correlation existed between methylation ofCpG motifs and SLE-DAI(p＜0.05).Expression of DNMT1 mRNA in patients withactive SLE and inactive SLE were lower than that in control group(p＜0.05),whilethere is no statistical difference between the active SLE and inactive SLE (p＞0.05),and no correlation existed between expression of DNMT1 mRNA and SLE-DAI(p＜0.05).There was no statistical difference in expression of DNMT3A mRNAbetween the active SLE,inactive SLE and normal controls.The expression ofDNMT3B mRNA was very low.There was no correlation between methylation ofCpG motifs and expression of DNMT1 mRNA (p＜0.05)in patients with SLE.Conclusions Hypomethylation of CpG motifs play an important role in thepathogenesis of SLE.Lower espression of DNMT1 mRNA may play a role inpathogenesis of SLE,which is not the exclusive regulation factor of methylation ofCpG motifs of SLE.PartⅡEffects of 5-azacytidine,estradiol and ultraviolet on the methylationof CpG motifs and expression of DNA Methyltransferasel Objective To investigate the effects of 5-azacytidine,estradiol and ultraviolet(254nm)on the methylation of CpG motifs and expression of DNAMethyltransferasel(DNMT1)mRNA in the patients with systemic lupuserythematosus(SLE)and normal controls.Methods The peripheral bloodlymphocytes isolated from 12 patients with SLE,and 11 normal individuals werestimulated with PHA for 1 day and then cultured an additional 3 day with or without5-azacytidine,estradiol and ultraviolet exposure respectively.The methylation of CpGmotifs was detected by the 5-Methylcytosine antibody and flow cytometry,and theexpression of DNMT1 mRNA of was analysed by real time RT-PCR.Results Aftertreatment with 5-azacytidine,Methylation of CpG motifs were lower in patients withSLE and the normal controls (p＜0.01),while there is no statistical difference in theexpression of DNMT1 mRNA in patients with SLE and the normal controls (p＞0.05).Nevertheless,there were no significant changes in the methylation of CpG motifs andexpression of DNMT1 mRNA in patients with SLE and normal controls before andafter the treatment with estradiol and ultraviolet exposure(p＞0.05).Conclusions Themethylation of CpG motifs was inhibited efficiently by 5-azacytidine,which effect maynot depend on the espression of DNMT1 mRNA.Estradiol and ultraviolet(254nm)may have no significant impact on methylation of CpG motifs and espression ofDNMT1 mRNA.PartⅢProkaryotic expression,purification and identification oftetrameric-protein of methyl-CpG-binding domainObjective To express,purify and identify tetrameric protein of methyl-CpG-bindingdomain in E.coli.Methods The recombinant plasmid 4xMBD-pET30b+ weretransformed into E.coli DHSa for clonal expansion and sequenced,then thetetrameric-protein were expressed in E.coli BL21 (DE3)under the induction of IPTG.Moreover,the expression product were purified by Ni-NTA,and were determined bySDS-PAGE and Western Blotting.Results The sequence analysis showed orientationwas right and was identical with the expectation.SDS-PAGE and Western Blotting demonstrated that the molecular weight of the tetrameric-protein was 46000,with theN-terminal His-tag and the C-terminal HA-tag.Conclusions The tetrameric-protein ofmethyl-CpG-binding domain was successfully expressed in E.coli and purified.Thisresults established a groundwork for the further researches of DNA methylation andits regulation factors.