Regulation Mechanism Of DNA Methylation And MiRNA On The Expression Of AlOX5AP In Peripheral Blood Of Patients With Ischemic Stroke

Background and Purpose:Ischemic stroke(IS)is a neurological disease caused by stenosis or obstruction of cerebral artery with the main clinical manifestations of ischemia,anoxia or necrosis in cerebral tissue of perfusion area.And it is characterized by high incidence,high disability and high mortality in China.The formation and development of atherosclerosis(AS)is one of the important pathophysiological mechanisms of IS.And inflammation is the key pathogenesis of AS.The pathway of 5-lipoxygenase activating protein/5-lipoxygenase(FLAP/5-LO)is a key metabolic pathway of leukotriene(LT),playing an important role in the biogenesis of LT.FLAP,encoded by ALOX5 AP gene,could combine the 5-LO to adjust the biogenesis of LT,activating the neutrophils and monocytes to increase the adhesion and permeability of the vascular internal wall,participating in the process of AS,and being closely related to IS.Str?m et al have reported that knockout of the ALOX5 AP gene in mice was associated with decreased LT production and amelioration of stroke damage.Xu et al have identified the correlation between the overexpression of ALOX5 AP and hypertensive stroke in rats.Our research group has found that the mRNA expression level of ALOX5 AP gene in the cases was significantly higher than that in controls by quantitative PCR.Therefore,we inferred that the mRNA expression level of ALOX5 AP gene in the cases was significantly higher than that in controls and it may also mediate the occurrence of IS by the involvement of inflammatory response in arterial wall.Epigenetics is a heritable change of gene expression without the alterations of gene sequences,providing new ways to reveal the regulation mechanism of gene expression.DNA methylation is an important form of epigenetic regulation.It is a process by which methyl groups are added to the cytosine in DNA molecule,forming the 5-methylcytosine.It is usually considered that DNA methylation could inhibit the expression of a gene by hindering the combination of transcription factors and DNA molecule.While the effect of DNA methylation of ALOX5 AP gene promoter on the expression of ALOX5 AP in peripheral blood of IS patients is not clear.In addition,CpG-SNP which was produced by the introduction or removal of cytosine-phosphate-guanine(CpG)dinucleotides could affect the expression level of a gene via allele-specific DNA methylation(ASM).To date,associated study between the promoter CpG-SNPs and DNA methylation in the ALOX5 AP gene has not been reported.MicroRNA is a small non-coding RNA molecule whose main function is to regulate gene expression by hindering the translation of specific mRNAs at the post transcriptional level.However,whether there existed some miRNAs influenced the expression level of ALOX5 AP gene in peripheral blood of IS patients hasn't been reported.In conclusion,we hypothesized that epigenetic mechanisms such as DNA methylation and miRNA regulation might have effect on the expression of ALOX5 AP gene in peripheral blood of IS patients.And it may mediate the occurrence of IS by the involvement of inflammatory response in arterial wall.The study will reveal the effect of DNA methylation and mi RNA on the expression of ALOX5 AP in peripheral blood of IS patients,providing a new theoretical basis for the genetic mechanism and prevention of the disease.Study Population:The study protocol was authorized by the Ethics Committee on Human Research of Zhengzhou University,the First People's Hospital of Zhengzhou,the First Affiliated Hospital of Henan University of Chinese Medicine and the First Affiliated Hospital of Zhengzhou University.And all the subjects were Henan Han population and signed the informed consent.1.The study population for examining the mRNA expression level of ALOX5APWe included 150 patients with IS(95 males and 55 females,mean age of 60.76�12.83 years)who were recruited from departments of neurology in the First People's Hospital of Zhengzhou and the First Affiliated Hospital of Zhengzhou University from April 2016 to July 2017 in the study.IS was diagnosed by the WHO diagnostic criteria of IS and confirmed by various examinations(medical history,physical signs,biochemical tests,CT or MRI).The control group consisted of 150 subjects(80 males and 70 females,mean age of 60.83 � 13.44 years)selected from the same demographic area.And they were all well matched by age and gender.2.The study population for examining the DNA methylation level of ALOX5APThe study population was the same as that examining the mRNA expression level of ALOX5 AP.3.The study population for examining the expression of plasma miRNAsWe included 50 patients with IS(30 males and 20 females,mean age of 62.42�13.04 years)who were recruited from departments of neurology in the First People's Hospital of Zhengzhou and the First Affiliated Hospital of Henan University of Chinese Medicine from April 2016 to July 2017 in the study.IS was diagnosed by the WHO diagnostic criteria of IS and confirmed by various examinations(medical history,physical signs,biochemical tests,CT or MRI).The control group consisted of 50 subjects(28 males and 22 females,mean age of 58.80�13.66 years)selected from the same demographic area.And they were all well matched by age and gender.Methods: 1.Total RNA was extracted using RNAprep Pure Blood Kit(Tiangen,Beijing,China)according to the manufacturer's instructions.Total RNA(300 ng for eachparticipant)was reversely transcribed into complementary DNA(cDNA)byFastKing RT Kit(Tiangen,Beijing,China)according to the manufacturer'sinstructions.The expression levels of ALOX5 AP gene were detected byquantitative PCR.2.Genomic DNA was extracted from peripheral blood samples with TIANampBlood DNA Kit(Tiangen,Beijing,China)according to the manufacturer'sinstructions.And the methylation levels of CpG sites located in the promoter ofALOX5AP were tested by MethylTarget sequencing.3.CpG-SNP rs4073259 was genotyped by Kompetitive Allele-Specific PCR(KASP)technology.Then we compared the differences of DNA methylation levels andmRNA expression levels of ALOX5 AP gene among different genotypes.4.The transcriptional activities of different alleles were detected by in vitrodual-luciferase assay.5.Plasma mi RNAs were extracted by Plasma miRNA isolation kit(Tiangen,Beijing,China)according to the manufacturer's instructions.And then themiRNAs were reversely transcribed.The expression levels of miR-335 andmiR-495 in peripheral blood plasma were detected by quantitative PCR.6.Dual-luciferase assay was designed to explore the combination of miR-335,miR-495 with the 3'UTR of ALOX5 AP gene.7.All statistical analyses were performed using the SPSS 21.0 package(SPSS Inc.,Chicago,IL,USA).Quantitative variables were expressed with mean�SD.TheStudent's t-test was used between two groups and the one-way ANOVA withBonferroni correction was used among three different groups.Pearson'schi-squared test was used to test for differences in qualitative variables.P valuesless than 0.05 were considered statistically significant.Results: 1.The mRNA expression level of ALOX5 AP in the cases was significantly higherthan that in controls(P<0.05).2.Compared with the controls,the DNA methylation level of ALOX5 AP genepromoter in the cases has no significant differences(P>0.05).Sex stratificationanalysis have also found no significantly differences between the two groups(P>0.05).3.CpG-SNP rs4073259 was associated with differential DNA methylation,revealingthe phenomenon of allele-specific methylation(ASM).And GG genotype carriersthat have a higher DNA methylation level also have a lower mRNA expressionlevel of ALOX5 AP.4.The relative luciferase activity produced by the G allele construct wassignificantly lower than that produced by the A allele construct,suggesting thatthe G allele with higher methylation level could down-regulate the geneexpression.5.The expression levels of plasma mi R-335 and miR-495 were significantly lowerin cases than those in controls(P<0.05).6.mi R-335 and miR-495 could regulate the expression of ALOX5 AP by combiningthe 3'UTR of the gene.Conclusions: 1.The mRNA expression level of ALOX5 AP in the cases was significantly higherthan that in controls(P<0.05).2.CpG-SNP rs4073259 located in the promoter of ALOX5 AP might affect theexpression level of the gene through allele-specific methylation and consequentlythe phenotype of the disease.3.The mRNA expression levels of miR-335 and miR-495 were significantly lowerthan those in controls(P<0.05).And they could regulate the expression level ofALOX5AP by combining the 3'UTR of the gene.