| Backgroud and Objective:Homocysteine(Hcy) is a kind of nonprotein amino acid formed during the metabolism of methionine in vivo.Physiological function of homocysteine is participating in the metabolism of one carbon unit. Abundant epidemiological evidence has demonstrated that the increasing concentration of Hcy in plasma can cause atherosclerotic vascular disease as an independent risk factor for atherosclerosis(As).Therefore,the intensive studies of mechanisms for Hcy-induced atherosclerosis may be the initial representation.In the past few years,the development of epigenetics has revealed many important mechanisms that environmental factors modulate gene expression.The acetylation and methylation modification of the chromatin and DNA sequence are most meaningfully regarded as main epigenetic patters.Hcy is an intermediate metabolite of methionine cycle,so abnormal elevated level of Hcy may affect DNA methylation,subsequently change gene expression and induce disease. Folic acid(FA), as an important micronutrient,is critical for maintaining genome stability. Folic acid is thought to markedly inhibit Hcy-induced atherosclerosis,at the same time FA is required for the synthesis of methionine and S-adenosylmethionine via a series of metablisms,the common methyl donor required for maintenance of DNA methylation. The 5,10-Methylenetetrahydrofolate reductase(MTHFR) is the key enzyme for Hcy down-regulation conversion,and acts as a critical juncture in folate metablism by catalyzing 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Deficiency of folate or a decrease of MTHFR activity induce the increasing concentration of Hcy in plasma.The putative MTHFR promoter does not have a TATA box but contains CpG islands and multiple potential Spl binding sites,this becomes structure base of MTHFR mRNA expression regulated by methylation. So we presume Hcy may by contraries affect methylation status in the promoter region of the MTHFR gene and subsequently change its gene expression.No reports showed what pathophysiological response will be induced by this effect. Main purpose of this study is to investigate the effect of homocysteine by clinic-relevant high concentrations on the methylation modification of MTHFR gene promoter and its mRNA expression in cultured human vascular smooth muscle cells(HVSMCs),in order to determine whether high Hcy can cause an injurious response of positive feedback by inducing aberrant methylation in the promoter region of the MTHFR gene,namely high Hcy→aberrant methylation in the promoter region of the MTHFR gene→MTHFR mRNA expression↓→MTHFR activity↓→more Hcy, at last result in progressive development in atherogenesis. At the same time, to investigate the intervention of folate is to justify hopes for novel preventive and therapeutic avenues.Material and methods:In this study HVSMCs from umbulical arteries using primary explant-method were isolated and cultured in DMEM/F12 medium supplemented with 20% fetal calf serum(FCS) at 37℃in a humidified 5% CO2 to 95% air atmosphere.All HVSMCs cultured were used between passage 3 and 7.Groups of experiment: HVSMCs were cultured in culture benchs.When cells were at subconfluence(85%～95%),they were preincubated in fresh serum-free medium for 24h,and then they were divided into following groups randomly at different concentrations of Hcy and folic acid. The SMCs were treated with clinic-relevant high homocysteine concentrations (0,30,100,200, 500,1000μmol/L) for 24h,48h,72h,or with 100μmol/L FA or FA / Hcy for 72h; the group of 0. 00μmol/L Hcy was the presence.The medium of all the cells incubated for 24h were replaced by fresh medium above.Cell genome DNA were extracted from different groups,and nested-touchdown-methylation-specific polymerase chain reaction (MS-PCR) was performed to catalyze the methylation pattern of the MTHFR promoter region. Total RNA were extracted by Trizol from different groups,and semiquantitative RT-PCR was used to detect the MTHFR mRNA expression.All data were summarized with mean±standard deviation. Results were analyzed by software package of SPSS13.0 .Statistical comparisons between groups were performed by the one-way ANOVA.Values of P＜0.05 were considered significant.Results: The homocysteine induced demethylation in the promoter region of MTHFR gene in all concentrations of Hcy and different period in cultured HVSMCs, the methylation pattern in the MTHFR gene displayed significant hypomethylation; and the MTHFR mRNA expression demonstrated obvious upregulation as well.The effect of 100μmol/L Hcy on the MTHFR mRNA expression was the most obvious,and very high homocysteine concentrations still increased obviously the MTHFR mRNA expression. Furthermore, Hcy(100μmol/L) increased the production of MTHFR mRNA significantly in a treating time-dependent manner and 72h was the most obvious.However, folic acid may inhibit demethylation Hcy-induced in the promoter region of MTHFR gene,and MTHFR mRNA expression was reduced significantly in FA+Hcy group comparing with Hcy group(P＜0.05).Difference between FA group and control group were not found.Conclusion: Our experiment suggested that the homocysteine can induce demethylation in the promoter region of MTHFR gene in HVSMCs and upregulates the MTHFR mRNA expression: but folic acid may inhibit this response to a certain extent.Since the MTHFR is the key enzyme for Hcy down-regulation conversion, the demethylation in its promoter region and the consequent upregulation of MTHFR mRNA expression induced by hyperhomocysteine should be a compensative response,not be a injurious one.The present study demonstrate the effect of environmental factors on the DNA methylation modification may not only be injurious,but also be compensative. Therapeutic strategy to intervene DNA methylation modification have been a perspective,so clarifing which gene were demethylated,which one were hypermethylated and which DNA methylation modification was injurious, which one was compensative, should be theoretical and practical significance to formulate therapeutic avenues. |
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