The Role And Mechanism Of DNA N~6-methyladenine Modification In Hypertension

Purpose:DNA methylation plays an important role in the pathogenesis of primaryhypertension.DNA N6-methyladenine modification(6mA)is present in human tissues as a novel adenine methylation.The aim of this study was to examine the changes of 6mA DNA methylation in patients with clinical primary hypertension,mice and rat hypertension models and human aortic smooth muscle cells(HASMC),to investigate its role in the pathogenesis of primary hypertension and to explore potential therapeutic targets for primary hypertension.Method:The study is divided into three parts.Clinical trials:213 patients with primary hypertension and 124 healthy controls were enrolled to detect changes in 6mA DNA methylation levels and to analyze their correlation with clinical indicators related to hypertension.In vivo experiments:establishment of animal models of hypertension in rats and mice,Immunohistochemistry staining analysis of 6mA DNA methylation patterns in animal models of hypertension and correlation of 6mA protein and methylation regulator protein demethylase histone H2A double oxidase alk homologue 1(ALKBH1),N6 adenosine-specific DNA methyltransferase 1(N6AMT1).Cell experiments:HASMC cells were cultured,and the expression changes of 6mA DNA methylation,ALKBH1,N6AMT1,and HIF1α-mediated pathways were analyzed using hypertension-stimulating factors angiotensin Ⅱ(Ang Ⅱ)and endothelin 1(ET1)to stimulate ALKBH1 expression,RNA interference(siRNA)technology to inhibit ALKBH1 expression,cell proliferation experiment,cell migration analysis,immunohistochemical staining technology,and chromatin immunoprecipitation technology,respectively.Result:Part I 6mA DNA methylation levels were significantly negatively correlated with systolic and/or diastolic blood pressure in patients with primary hypertension(r=0.157,0.171;p=0.022,0.012).Post-group analysis according to the hypertension well-treated group(n=120),the poorly-treated group(n=93)and healthy controls(n=124)found that the level of leukocyte 6mA methylation was significantly lower in poorly-treated hypertensive patients than in healthy controls and the well-treated hypertensive group(p<0.05).After the treatment effectively controlled blood pressure,the 6mA methylation level returned to normal,and there was no difference between the 6mA DNA methylation level of the group with good hypertension treatment and the normal control group.Analysis of the correlation between medical history and 6mA DNA methylation levels revealed that patients with low 6mA DNA methylation usually had a long history of hypertension(r=-0.267,p<0.05).Linear regression analysis showed that after correcting for age and gender,6mA DNA methylation levels were negatively correlated with systolic blood pressure(SBP),diastolic blood pressure(DBP),total cholesterol(TC),homocysteine(HCY),triglycerides(TG)and low-density lipoprotein(LDL)levels and positively correlated with high-density lipoprotein(HDL)levels(r=-0.238,-0.219,-0.291,-0.391,-0.211,-0.187,0.315;p<0.05).Part Ⅱ 6mA DNA methylation levels were also decreased in mice and rat hypertension models.In hypertensive mice and rat models,6mA protein was significantly decreased in aortic vascular smooth muscle cells(VSMCs)of mice and rats in the hypertensive group compared to the control group.The demethylation enzyme ALKBH1 up-regulates protein expression in vascular smooth muscle cells of hypertensive mice or rats,and ALKBH1 is negatively correlated with 6mA protein expression.Methyltransferase N6AMT1 expression was not correlated with 6mA protein expression.Part Ⅲ Treatment of HASMC cells with Ang Ⅱ and ET-1 revealed dose-dependent upregulation of ALKBH 1 but not N6AMT1,decreased 6mA DNA methylation levels,and negative correlation between 6mA protein expression and ALKBH1 protein expression.When siRNA knockdown of ALKBH1 expression was followed by inhibition of AngⅡ and ET1 induction,both ALKBH1 expression was decreased,while 6mA DNA methylation levels were increased.After ALKBH 1 knockdown,expression of contractile phenotype markers a-smooth muscle actin(α-SMA)and calcium modulating protein(CALPONIN)was increased and proliferative marker bone bridge protein(OPN)was decreased.Downregulation of ALKBH 1 inhibited angiotensin Ⅱ-induced changes in vascular smooth muscle cells from contractile to proliferative phenotype.EdU cell proliferation assay analysis showed that angiotensin Ⅱ-induced cell proliferation was significantly inhibited after knockdown of ALKBH 1,cell migration assay and scratching assay showed that down-regulation of ALKBH1 reduced the number and distance of migrating vascular smooth muscle cells,and ALKBH1 maintained Ang Ⅱ-induced vascular remodeling.The third part of the study suppressed the elevated HIF1α levels by knocking down ALKBH1,and bioinformatics analysis revealed three 6mA peaks around the human HIF1α gene that increased with knockdown of ALKBH1 expression.Microarray analysis with 6mA antibodies confirmed that 6mA modifies all three peaks,and that methylation of the first peak can be inhibited by Ang Ⅱ stimulation and further corrected by knocking down ALKBH1 expression.Conclusion:ALKBHI-6mA regulation has a potential epigenetic role in the pathogenesis of hypertension.Levels of 6mA DNA methylation in peripheral blood leukocytes may serve as potential biomarkers of hypertension.The dynamic changes in 6mA DNA levels are controlled by ALKBH1.Inhibition of ALKBH1 expression in vascular smooth muscle cells inhibits AngⅡ-induced vascular smooth muscle type transformation,proliferation,and migration via the HIF1 α-dependent pathway.ALKBH1 is a key molecule mediating the interaction of 6mA DNA methylation level modifications with HIF1α activity during vascular remodeling and may be a novel therapeutic target for the inhibition of hypertension.