| Acetaldehyde dehydrogenase 2 (ALDH2) is a kind of nuclear encoding enzyme, and its main effect is to catalyze energy-metabolism-related aldehyde products or substrates into specific organic acid in mitochondrial matrix. Researches in recent years illustrated high expression of ALDH2 in myocardium, and proved ALDH2 myocardium protection effect in various myocardial damage models. In the setting of myocardium ischemia, by detoxifying aldehyde 4-Hydroxynonenal (4-HNE),ALDH2 can block 4-HNE related reactive oxygen species axis, ameliorate oxidative stress response, abrogate HSP70/JNK/p53 mediated downstream myocardial apoptosis pathways and hence play protective role in myocardium. On the other hand, down-regulation of ALDH2 level was proved in ischemia myocardium, and exogenous ALDH2 replenishment could alleviate myocardium ischemia damage. Epigenetics refers to as epigenome, and it is interpreted as epigenetic changes that does not change gene sequence structure including DNA methylation, histone modifications and non-coding RNAs such as microRNAs. In Epigenetics factors, DNA Methylation can influence gene transcription by transforming cytosine in CpG site into 5-methyl-cytosine with adding methyl groups. Generally, high methylation level suppresses gene transcription. Moreover, rerearchers found more and more clinical cardiovascular disease can be explained and further understood by epigenetics mechanisms.Our previous research showed up-regulation of microRNA34a in ischemia myocardium, and specific binding of microRNA34a and ALDH2 mRNA can inhibit ALDH2 translation. This result provide promising insights into epigenetics mechanisms of ALDH2 ischemia-related down-regulation, while such phenomenon cannot be fully explained by microRNA alone. Accordingly, it is reasonable to continue exploring epigenetics mechanisms of ALDH2 ischemia-related down-regulation, and DNA methylation fits quite right.We hence hypothesized, in the setting off myocardial ischemia, ischemia and hypoxia stimulation might result in changes of DNA methylation level in ALDH2 promoter related sequences, and such changes further brought about ALDH2 down-regulation. To address this issue, our preliminary experiment was focused on comparison of test Effectiveness on Acetaldehyde dehydrogenase 2 (ALDH2) specific low-methylated region between MassARRAY quantitative methylation analysis and Bisulfite sequencing PCR(BSP). The comparison assured preferable method for specific ALDH2 gene sequence. (PartⅠ)On the base of Part I, we conducted myocardium infarction models with Sprague Dawley Rattus. Various examinations were carried out to detect DNA methylation related data. In brief, this study aims to explore potential DNA methylation mechanism of ALDH2 ischemia-related down-regulation, find ALDH2 gene sequence with aberrant methylation level and provide new targets and insights for clinical treatment.ObjectiveBy comparing the test effectiveness between MassARRAY quantitative methylation analysis and Bisulfite sequencing PCR(BSP), we try to determine an effective method to examine the DNA methylation level of low-methylated region(LMR).MethodTen male Sprague-Dawley rats were assigned into two groups, each group contained five rats. Rats of experiment group were performed left anterior descending (LAD) artery ligation surgery for myocardial infarction (MI) model. Sham group and experiment group were harvested for MI border zone myocardial tissue after 7 days. After the DNA extraction, MassARRAY quantitative methylation analysis and Bisulfite sequencing PCR were performed to test the DNA methylation of a LMR sequence in ALDH2 gene, respectively.Result BSP identified 12 CpG sites in target LMR, and the test result of BSP showed the baseline methylation of all 12 CpG sites were 0 in both groups. MassARRAY quantitative methylation analysis only identified 10 CpG sites, while its result indicated there was low DNA methylation level in both group, what’s more, compared with sham group, DNA methylation level of No.5 and No.6 CpG site in experiment group were higher, while No.7 statistically lower(P<0.05).ConclusionCompared with BSP, MassARRAY quantitative methylation analysis has higher effectiveness and accuracy in testing DNA methylation level for low-methylated region. To maintain accuracy for DNA methylation examination in ALDH2 promoterObjectiveBy comparing the test effectiveness between MassARRAY quantitative methylation analysis and Bisulfite sequencing PCR(BSP), we try to determine an effective method to examine the DNA methylation level of low-methylated region(LMR).MethodTen male Sprague-Dawley rats were assigned into two groups, each group contained five rats. Rats of experiment group were performed left anterior descending (LAD) artery ligation surgery for myocardial infarction (MI) model. Sham group and experiment group were harvested for MI border zone myocardial tissue after 7 days. After the DNA extraction, MassARRAY quantitative methylation analysis and Bisulfite sequencing PCR were performed to test the DNA methylation of a LMR sequence in ALDH2 gene, respectively.Result BSP identified 12 CpG sites in target LMR, and the test result of BSP showed the baseline methylation of all 12 CpG sites were 0 in both groups. MassARRAY quantitative methylation analysis only identified 10 CpG sites, while its result indicated there was low DNA methylation level in both group, what’s more, compared with sham group, DNA methylation level of No.5 and No.6 CpG site in experiment group were higher, while No.7 statistically lower(P<0.05).ConclusionCompared with BSP, MassARRAY quantitative methylation analysis has higher effectiveness and accuracy in testing DNA methylation level for low-methylated region. To maintain accuracy for DNA methylation examination in ALDH2 promoterPart I:Comparison of Test Effectiveness on Acetaldehyde dehydrogenase 2 (ALDH2) specific low-methylated region between MassARRAY quantitative methylation analysis and Bisulfite sequencing PCR related sequence, MassARRAY was suggested as an essential tool for ALDH2 methylation test.ObjectiveAldehyde dehydrogenase 2(ALDH2) plays a critical role in myocardial protection against ischemia, but there is no evidence to explain why ALDH2 expression of myocardial cells decreases during ischemia. DNA methylation is a epigenetic way to alter the transcription level of genes. We hypothesized that methylation level of ALDH2 gene could affect ALDH2 expression during myocardial infarction.MethodSprague-Dawley Rats were randomly assigned into four groups for Myocardial infarction (MI) model. MI border region tissues were harvested after rat MI model, at different time point(1st week,2nd week,3rd week) respectively. Bisulfite sequencing PCR (BSP) was performed to detect the methylation levels of ALDH2 core promoter. Sequenom MassARRAY platform was performed to examine the methylation levels of ALDH2 promoter upstream sequence. ALDH2 protein expression was assayed by western blot. ResultWestern blot indicated that ALDH2 protein level decreases after MI.DNA methylation has no effect on rat ALDH2 core promoter in the progress of myocardial ischemia injury, while the methylation level of CpG sites in ALDH2 promoter upstream sequence was significantly higher n MI experiment groups(CpG1,CpG2,CpG7,P<0.01), however, no statistical differences were shown among MI experiment groups.ConclusionAberrant hypermethylation of CpG sites in ALDH2 promoter upstream sequence isObjectiveAldehyde dehydrogenase 2(ALDH2) plays a critical role in myocardial protection against ischemia, but there is no evidence to explain why ALDH2 expression of myocardial cells decreases during ischemia. DNA methylation is a epigenetic way to alter the transcription level of genes. We hypothesized that methylation level of ALDH2 gene could affect ALDH2 expression during myocardial infarction.MethodSprague-Dawley Rats were randomly assigned into four groups for Myocardial infarction (MI) model. MI border region tissues were harvested after rat MI model, at different time point(1st week,2nd week,3rd week) respectively. Bisulfite sequencing PCR (BSP) was performed to detect the methylation levels of ALDH2 core promoter. Sequenom MassARRAY platform was performed to examine the methylation levels of ALDH2 promoter upstream sequence. ALDH2 protein expression was assayed by western blot. ResultWestern blot indicated that ALDH2 protein level decreases after MI.DNA methylation has no effect on rat ALDH2 core promoter in the progress of myocardial ischemia injury, while the methylation level of CpG sites in ALDH2 promoter upstream sequence was significantly higher n MI experiment groups(CpG1,CpG2,CpG7,P<0.01), however, no statistical differences were shown among MI experiment groups.ConclusionAberrant hypermethylation of CpG sites in ALDH2 promoter upstream sequence isPart II:DNA methylation level of AldehydeDehydrogenase 2 Promoter Upstream Sequence in Rats with Experimental Myocardial Infarction associated with myocardial ischemia injury and this suggests a potential mechanism to explain the decrease of ALDH2 gene expression during MI. |
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