Association Study Between CELSR1Polymorphisms And Ischemic Stroke In Chinese Han Population

Background and ObjectivesUsing GWAS analysis, Yoshiji Yamada group identified CELSR1as asusceptibility gene for ischemic stroke（IS） in Japanese individuals. CELSR1is a keycomponent of the non-canonical Wnt/PCP pathway, and the non-canonical Wnt/PCPpathway was recently found to play a critical role in angiogenesis. The present studywas designed to examine whether CELSR1variants are associated with IS in ChineseHan population. We transfected small interfering RNA (siRNA) to mute the CELSR1expression of in human aortic endothelial cells （HAECs） by liposomes instanttransfection to explore the effects of inhibition of CELSR1expression on proliferation，migration of HAECs.Methods1.We carried out a case-control association study using a total of515cases and533controls.The genotypes of two single nucleotide polymorphisms, rs6007897andrs4044210of CELSR1, were evaluated by polymerase chain reaction-restrictionfragment length polymorphism(PCR-RFLP)and direct DNA sequencing. We assessedtheir allele, genotype and haplotype associations with IS. The analysis was adjusted forconfounding variables and stratified by stroke etiology.2.We transfected small interfering RNA (siRNA) to mute the CELSR1expressionof in HAECs by liposomes instant transfection. After siRNA was transfected intoHAECs， the expression level of CELSR1was determined by Western blot,Immunohistochemistry and qRT-PCR， the cell proliferation and migration wasdetected by MTT assay and erasion trace test respectively.Results1.The results showed that, rs6007897of CELSR1(P <0.05) and the rs4044210of CELSR1(P <0.05) were associated with IS in terms of both genotype distributionand allele frequency. The haplotype analysis demonstrated that the frequency of theminor haplotype G–G was significantly higher when compared with that of the major haplotype A-A, in subjects with IS than in controls. Moreover, the multivariate analysisshowed that both rs4044210and rs6007897were significantly associated with subjectswith large artery arteriosclerosis.2.CELSR1expression was detected in human umbilical vein endothelial cells(EA.hy926), HAECs and human umbilical vein cells（HUVECs）. The maxmiuminhibitory effects of CELSR1-siRNA transfection on CELSR1expression was at48h.The CELSR1gene was knocked down by RNAi,with silence rate of88.2%. AftersiRNA was transfected into HAECs, the growth was significantly inhibited about65.6%and the migration was also significantly inhibited.Conclusion1.the present study has proven that CELSR1is a susceptibility gene for IS inChinese Han population.2.CELSR1gene in HAECs can be knocked down effectively by RNAi,CELSR1-siRNA transfection is able to inhibit the growth and migration of HAECs invitro.