Association And Function Of CAMTA1 Promoter Methylation In Ischemic Stroke

BackgroundIschemic stroke(IS)refers to a series of clinical diseases caused by ischemia,hypoxia or necrosis of brain tissue in the perfusion area due to the stenosis or obstruction of cerebral artery.Stroke is the leading cause of death and disability in China,which brings heavy health and economic burden to individuals and families.The formation and development of atherosclerosis(AS)is an important pathophysiological basis of IS.AS is a disease characterized by fibrosis and proliferation of vascular wall,lipid accumulation,chronic inflammation and immune disorder.Macrophage inflammation induced by oxidized-low density lipoprotein(ox-LDL)plays a key role in the occurrence and the development of AS.When macrophages phagocytize ox-LDL deposited under the artery endothelium,it can stimulate cells to produce a large number of chemokines and inflammatory factors,thus aggravating vascular inflammatory response.DNA methylation is a chemical modification of DNA.It is that the methyl group of S-adenosylmethionine(SAM)combines to the 5'carbon site of genomic CpG dinucleotide cytosine to form 5-methylcytosine(5-mC)under the action of DNA methyltransferase(DNMT).Methylation modification of DNA specific sites can change DNA stability,DNA conformation,chromatin structure and interaction between DNA and protein,thus regulating gene expression and participating in the regulation of metabolic pathway.Using Illumina 850K methylation chip technology and MethylTarget sequencing technique,we found that the methylation level of CpG island in the promoter region of CAMTA1 in patients with ischemic stroke was significantly higher than that in healthy controls(P<0.05).Calmodulin binding transcription activator 1(CAMTA1)belongs to the family of calmodulin binding transcription activators,which contains IQ motifs and may respond to calcium signal pathway through direct binding to calmodulin.Genome-wide association and large-scale candidate gene research have found that mutations in CAMTA1 gene are associated with early-onset coronary artery disease.It has been reported that CAMTA1 may be involved in the positive regulation of calcineurin/nuclear factor of activated T cell(CaN/NFAT)signal pathway,and then participate in cellular immune regulation.To sum up,the following hypothesis is proposed:the methylation level of CAMTA1 promoter increases in patients with ischemic stroke,which participates in the regulation of CAMTA1 gene expression,and participates in the pathological process of IS by affecting the inflammatory response of cells.In this study,we will study the effect of DNA methylation on CAMTA1 gene expression,and further study the functional mechanism of CAMTA1 protein involved in IS.These can provide experimental basis for exploring the genetic mechanism of IS and exploring effective prevention and treatment methods of IS.Materials and methodsThe first part:study on the relationship between methylation in the promoter region of CAMTA1 and ischemic stroke1.Study population:the case group selected 110 IS patients who were hospitalized in the First Affiliated Hospital of Henan University of Chinese Medicine and the First People's Hospital of Zhengzhou from October 2017 to March 2019.The control group selected 110 healthy individuals whose sex and age matched those of the case group in the same period.The study protocol was authorized by the Ethics Committee on Human Research of Zhengzhou University.2.DNA was extracted by blood genome extraction kit.The methylation level of CpG island in the promoter region of CAMTA1 was detected and analyzed by MethylTarget sequencing.3.The total RNA of peripheral blood was extracted by blood total RNA extraction kit.The mRNA expression level of CAMTA1 gene was analyzed by qPCR.4.Peripheral blood mononuclear cells were isolated by Ficoll density gradient separation,and the expression of CAMTA1 was detected by qPCR and Western blot.The second part:the effect of CAMTA1 on the expression of inflammatory factors in THP-1-derived macrophages induced by ox-LDL1.THP-1 macrophages were treated with 80 ?g/mL ox-LDL for 0,6,12,24,48 h respectively.The contents of IL-6,TNF-? and IL-1? in the supernatant of THP-1 macrophages were detected by ELISA.2.THP-1 macrophages were transfected with siRNA-CAMTA1,and the contents of IL-6,TNF-? and IL-1? in the supernatant were detected by ELISA.3.The CAMTA1 overexpression plasmid was used to transfect THP-1 macrophage,and the contents of IL-6,TNF-?,IL-1? in the supernatant were detected by ELISA.Results1.The average methylation level of CpG island in the promoter region of CAMTA1 gene in IS group was significantly higher than that in the control group(P<0.05).There was a significant difference in the methylation level of CpG 16 and CpG21 between the case group and the control group(P<0.05).There was no significant difference between the case group and the control group after gender stratification analysis(P>0.05).2.The mRNA expression level of CAMTA1 in IS group was significantly lower than that in the control group(P<0.01).3.There was a negative correlation between the methylation of CAMTA1 promoter and the mRNA expression of CAMTA1,which was statistically significant(P<0.0001).4.The mRNA and protein expression level of CAMTA1 in mononuclear cells of IS group was significantly lower than that of control group(P<0.01).5.The secretions of IL-6,TNF-? and IL-1? were the most significant when THP-1 macrophages were treated with ox-LDL for 24 h(P<0.01).6.The concentration of IL-6,TNF-? and IL-1? in the supernatant of THP-1 macrophages decreased significantly when CAMTA1 was knocked down(P<0.01).7.The concentration of IL-6 and TNF-? in the supernatant of THP-1 macrophages increased significantly when CAMTA1 was overexpressed(P<0.01).Conclusion1.The hypermethylation in the promoter region of CAMTA1 gene inhibits the mRNA expression of CAMTA1,which leads to the decrease of CAMTA1 protein in patients with ischemic stroke.2.In the model of AS inflammation induced by ox-LDL,knocking down CAMTA1 can inhibit the secretion of IL-6,TNF-? and IL-1?.3.In the model of AS inflammation induced by ox-LDL,overexpression of CAMTA1 can promote the secretion of IL-6 and TNF-?.