Association And Functional Study Of C14orf119 Gene 3’UTR Snps And Ischemic Stroke Susceptibility In Han Chinese

OBJECTIVE:This study aimed to explore the C14orf119 mRNA expression level in human peripheral blood samples,the association of C14orf119 gene3’untranslated region（3’UTR）single nucleotide polymorphisms（SNPs）including rs12400（C>T）and rs6736（A>T）,and the related clinical risk factors with ischemic stroke（IS）susceptibility in Han Chinese,as well as to preliminarily explore the potential function of C14orf119 gene 3’UTR SNP rs6736（A>T）to modify C14orf119 gene binding to miRNA-7-1.METHODS:Using case-control study,this study enrolled 774 patients with IS and 793 healthy controls.Using quantitative real-time polymerase chain reaction（qRT-PCR）,C14orf119 gene mRNA expression level was detected in human peripheral blood.Using Sequenom MassARRAY platform for genotyping of the C14orf119 gene rs12400 and rs6736 polymorphisms.Using dual luciferase reporter gene assay,the function of C14orf119 gene 3’UTR SNP rs6736 to modify C14orf119 gene binding to miRNA-7-1 was explored in human embryonic kidney293T（HEK293T）cells.RESULTS:1.Expression level of C14orf119 gene:The C14orf119 gene mRNA increasingly expressed in peripheral blood samples of IS cases compared to that of controls,with statistically significance（t=2.235,P=0.030）.2.Association between C14orf119 gene 3’UTR polymorphisms and IS:（1）Hardy-Weinberg Equilibrium（HWE）test:The genotype frequency distribution of C14orf119 gene SNPs rs12400(P_HWE=0.510)and rs6736(P_HWE=1.000)in the control group were both in line with HWE.（2）Association analysis between C14orf119 gene 3’UTR polymorphisms and IS susceptibility:The genotype frequency distribution of rs6736 in IS cases and controls were statistically significantly different（overall:?²=6.402,P=0.041;female:?²=6.196,P=0.045）,but there was no statistically significant difference existed in the genotype frequency distribution of rs12400 between IS cases and controls.Multi-variate logistic regression analysis showed that,the association between C14orf119 gene rs6736 and IS susceptibility was statistically significant[dominant model:OR（95%CI）=0.755（0.606?0.940）,P=0.012;co-dominant 1model:OR（95%CI）=1.340（1.060?1.694）,P=0.014;allelic model:OR（95%CI）=0.865（0.751?0.997）,P=0.046];after adjustment of sex and age,the statistical significance remained between C14orf119 gene rs6736 and IS susceptibility[dominant model:OR（95%CI）=0.758（0.609?0.945）,P=0.014;co-dominant 1model:OR（95%CI）=1.329（1.051?1.681）,P=0.018];but there was no statistical significance existed between C14orf119 gene rs12400 and IS risk（all of the genetic models:P>0.05）.（3）Correlation analysis between C14orf119 gene polymorphisms and IS-related clinical risk factors:1)Overall:C14orf119 gene rs12400 and rs6736polymorphisms were not significantly correlated with clinical index including blood pressure（SBP,DBP）,blood glucose（FPG,F2hPG）,blood lipids（TC,TG,HDL,LDL,VLDL,APOA1,APOB）,blood coagulation（PLA,PT,INR,PTA,APTT,TT,FBG）and others（CRP,D-D,HCY,UA）in IS patients（All P>0.050）.2)Stratified analysis by gender:In males,C14orf119 gene rs6736 polymorphism was significantly negatively correlated with fibrinogen（FBG）in male patients with IS[additive model:β（95%CI）=-0.148（-0.290?-0.005）,P=0.043;β_adj（95%CI）=-0.143（-0.285?-0.002）,P_adj=0.047],while C14orf119 gene rs12400polymorphism was not significantly correlated with clinical index including blood pressure（SBP,DBP）,blood glucose（FPG,F2hPG）,blood lipids（TC,TG,HDL,LDL,VLDL,APOA1,APOB）,blood coagulation（PLA,PT,INR,PTA,APTT,TT,FBG）and others（CRP,D-D,HCY,UA）in male patients with IS（All P>0.050）.In females,C14orf119 gene rs12400 polymorphism was significantly negatively correlated with postprandial 2h blood glucose（F2hPG）[recessive model:β（95%CI）=-6.306（-11.060?-1.556）,P=0.012;β_adj（95%CI）=-6.701（-11.580?-1.820）,P_adj=0.010]and thrombin time（TT）[recessive model:β（95%CI）=-1.845（-3.086?-0.604）,P=0.004;β_adj（95%CI）=-1.838（-3.081?-0.595）,P_adj=0.004]in female patients with IS,while C14orf119 gene rs6736polymorphism was not significantly correlated with clinical index including blood pressure（SBP,DBP）,blood glucose（FPG,F2hPG）,blood lipids（TC,TG,HDL,LDL,VLDL,APOA1,APOB）,blood coagulation（PLA,PT,INR,PTA,APTT,TT,FBG）and others（CRP,D-D,HCY,UA）in female patients with IS（All P>0.050）;3.The dual luciferase reporter gene assay:The relative fluorescence intensity of the C14orf119 gene rs6736 wild-type（3’UTR-WT+miRNA-7-1）group significantly decreased,compared to that of the blank control（3’UTR-NC+miRNA-7-1）group（P<0.001）.The relative fluorescence intensity of the C14orf119 gene rs6736 mutant-type（3’UTR-MU+miRNA-7-1）group was not significantly different from the blank control group（P=0.703）.While the allele of C14orf119 gene rs6736 polymorphism is T,C14orf119 gene will bind to miRNA-7-1 with significant decrease in relative fluorescence intensity appearing.CONCLUSIONS:1.Peripheral blood mRNA expression of C14orf119 gene may be associated with the onset of IS.2.The C14orf119 gene rs6736 polymorphism may influence the occurrence of IS in Han Chinese,and the mutation of rs6736 could modify C14orf119 gene binding to miRNA-7-1.3.The C14orf119 gene SNPs may be involved in coagulation-related pathological processes in male patients with IS,and may affect blood glucose metabolism and coagulation in female patients with IS.