The Effect Of DNA Methylation On The Gene Of LRP1 And The Role In The Pathogenesy Of Coronary Artery Disease

Chapter one:The age-related hypermethylation of the promoter of LRP1 gene in the coronary heart disease patientBackgroundThe low density lipoprotein receptor-related protein 1(LRP1)is a important receptor which involves in the development of artherosclerosis. The new evidence shows that the LRP1 may play the dural role in the artherosclerosis include the protective and destructive effects.The regulation mechanism of LRP1 is still not clear.There were some clues indicated that modification in DNA methylation induced the LRP1 expression silence in some carcinogenesis,but whether this modification happens in the atherosclerosis has not been reported.ObjectivesTo investigate the differences of the LRP1 gene DNA methylation status between the normal and CHD patients,the relationship between the change of DNA methylation of LRP1 gene,age and the LRP1 expression levels.MethodsThe 112 coronary heart disease patients and 110 controls were included in this cross-sectional study.The DNA were extracted from the white blood cell and the DNA methylation status of LRP1 gene were studied using the bisulfite DNA sequencing,as well as the LRP1 mRNA and protein expression level were measured by RT-PCR and The dates were analysis using the SPSS 11.0 soft to find the relationship between the age,the LRP1 expression level and the methylation levels.ResultsThere was no significant difference of the LRP1 mRNA,protein expression levels and the average methylation levels of the promoter of the LRP1 gene between the young groups in normal and CHD patients; but LRP1 mRNA and protein expression levels lower between the old CHD patients and the controls(age＞50 years),the average methylation levels of the promoter of the LRP1 gene is higher in the old CHD patient than in the normal(P＜0.05).ConclusionIn the CHD patients,there exist hypermethylation in the promoter of LRP1 gene,which may results in the LRP1 expression change with age. Chapter two DNA hypomethylation and TNF-βinduced high expression of LRP1 in mononuclear cellsBackground:The LRP1 has been reported to express in high level in human mononuclear cells but the regulation mechanism of it still not very clear.There were some new evidences showed that the DNA methylation involved in the regulatory mechanism of LRP1 gene expression.In order to understand the effect of the DNA methylation on the LRP gene expression,we test these hypotheses by using the ox-LDL and the DNA methylation suppressor to interfere in the cultured mononuclear cells.Objective:To observe the effect of the DNA methylation inhibitor 5-aza-2'-deoxycytidine on the LRP1 expression in the mononuclear cells, and the effect of TNF-βcombined with DNA methylation suppressor 5-aza-2'-deoxycytidine on the expression of the LRP1.Methods:the monocytes which isolated from peripheral blood of normal donors were incubated and were intervened with 5-aza-2'-deoxycytidine or combined with the different concentration TNF-β,the LRP1 protein expression levels was measured by immunoblotting and the mRNA level of LRP1 was measured by reverse transcription polymerase chain reaction(RT-PCR).The difference of LRP1 gene expression levels between the blank and the intervention group cells were analysed using by SPSS software. Results:The methylation inhibitors 5-aza-2'-deoxycytidine promote monocytes expressing the LRP1 singly slightly in vitro,the TNF-βand the 5-aza-2'-deoxycytidine combined intervention induced higher LRP1 expression level.Conclusions:Intervention with 5-aza-2'-deoxycytidine induced the mononuclear macrophage cells highly expressing LRP1 in vitro,this effect was enhanced when the intervention was combined with the TNF-β. This result refers to that the DNA methylation involves in the regulatory mechanism of the LRP1. Chapter three:Effects of DNA hypomethylation on the expression of LRP1 and the proliferation/migration of VSMCBackground LRP1 which serves as lipoprotein and signal receptor has important.roles in the VSMC proliferation/ migration.Some new evidences have shown that DNA methylation may have effects on the LRP1 gene expression in the tumor cells,but whether it works in the normal cells such as VSMC is still not clear,and also it has not been reported that whether DNA methylation can affect VSMC proliferation.Objective This study is aimed to explore effects of DNA methylation inhibitor 5-aza-2'-deoxycytidine on the LRP1 expression and the proliferation/migration of VSMC.Methods VSMC cell lines were cultured by enzymatic dissociation methods.Subcultures between 9 and 16(p4-6)were used in the study. PDGF—BB was used to stimulate VSMC proliferation.The alive cell counting by trypan blue dye staining and tetrazolium dye-reduction assay(MTT)were used to determine the proliferation at different 5-aza-2'-deoxycytidine concentration(0,0.01,0.1,1,10μmol/L),the same methods were used to do with 10μmol/L PDGF—BB combination with 5-aza-2'-deoxycytidine.LRP1 protein was detected by the method of immunocytochemistry.The level of LRP1 mRNA was assessed by the method of RT-PCR. Results The PDGF—BB induced Proliferation/migration/ of VSMC were decreased with the increase of 5-aza-2'-deoxycytidine concentration.,a special inhibitor of DNA methylation.The increase of the expression in LRP1 was dependent on the dose of 5-aza-2'-deoxycytidine.Lactoferrin markedly reduced this effect of 5-aza-2'-deoxycytidineConclusion 5-aza-2'-deoxycytidine could inhibit VSMC Proliferation/migration.5-aza-2'-deoxycytidine could induce the expression of LRP1.Inducing LRP1 expression was one of the mechanisms responsible for 5-aza-2'-deoxycytidine inhibiting VSMC Proliferation/migration.