| Objective:To detect the expression of ARL6IP5 gene mRNA and protein in peripheral mononuclear cells(PBMCs)and plasma of patients with ischemic stroke(IS),and analyze the expression levels in patients with post-stroke cognitive impairment(PSCI)and post-stroke non-cognitive impairment(PSNCI).To identify the relationship of rs7038G/T SNP of ARL6IP5 gene in IS and PSCI by detecting the 3'-UTR rs7038G/T allele and genotype distribution of ARL6IP5 gene,and to discuss the correlation between ARL6IP5 genotype and ARL6IP5 gene transcription and translation.Methods:410 cases of IS patients(IS group)and 239 cases of healthy persons(healthy control group)were enrolled in the affiliated hospital of North Sichuan Medical College.Some of the patients with IS were divided into PSCI group(n=198)and PSNCI group(n=191)according to MMSE score.The peripheral venous blood was collected,total RNA were extracted by Trizol,and the plasma were centrifuged.The mRNA relative expression levels of ARL6IP5 gene in PBMCs and the protein expression levels of ARL6IP5 gene in plasma were detected through Real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)and Enzyme linked immunosorbent assay(ELISA)respectedly.The rs7038G/T polymorphism in ARL6IP5 gene was genotyped by Restriction fragment length polymorphism-Polymerse Chain Reaction(RFLP-PCR)and identified through direct sequencing.The genotype and allele frequencies of ARL6IP5gene rs7038G/T polymorphisms between the IS,healthy control,PSCI and PSNCI group were evaluated by the chi-square test or Fisher exact test.The risk association between the genotype and IS and PSCI was estimated by multivariate logistic regression analyses after adjusting by gender and age status.P value<0.05 was taken as the level for statistical significance.The relative risk associated with genotype and allele was analyzed by an odds ratio(OR)with a 95%confidence interval(CI).Results:(1)Logistic regression analysis showed that the percentage of neutrophil,platelet,triglyceride and creatinine in IS group and healthy control group were statistically significant(P<0.05).Those were no significant differences in leukocyte count,high-sensitive C-reactive protein,homocysteine and high density lipoprotein between the two groups(P>0.05).Multivariate logistic regression analysis showed that there were significant differences between PSCI group and PSNCI group in the degree of education,ability of daily living(ADL)assessment and history of heart disease(P<0.05),there were no significant difference in age,sex,smoking history,drinking history,multiple infarction,TOAST classification and neutrophil percentage(P>0.05).(2)120 cases of IS group and 60 cases of healthy control group were randomly selected for RT-qPCR and Elisa test.The relative expression of mRNA in IS group and healthy control group was 1.3382±1.2166,1.9863±1.4679,respectedly(P=0.001).And the relative expression of mRNA in PSCI and PSNCI group was 1.6991±1.4210 and 1.0914±0.9904,respectedly(P=0.004).The expression of plasma protein in IS and healthy control group was 3.7298±1.3441 and 2.7057±1.0141(P=0.001).And the expression of plasma protein was 3.3340±1.4727 in PSCI group and 4.0597±1.1494 in PSNCI group(P=0.045).(3)The single nucleotide polymorphisms(SNPs)of ARL6IP5 gene rs7038G/T were analyzed in 410 IS patients and 239 healthy controls.The Hardy-Weinberg genetic equilibrium test was carried out by?~2 test of fitness degree in healthy control group.The results(P=0.064)showed that the distribution of rs7038G/T bases in healthy control group was in accordance with Hardy-Weinberg genetic balance and showed good population representation.The T allele frequencies of rs7038G/T polymorphism in IS group and healthy control group were 60.4%and 70.3%,respectively.G allele frequencies were 39.6%and 29.7%.The frequency of G allele of rs7038G/T polymorphism in healthy control group was significantly lower than that in IS group(P<0.0001,OR=0.64,95%CI=0.52–0.82).Co-dominant genetic model(heterozygous model and homozygous model),dominant genetic model and recessive genetic model were used to analyze the risk relationship between genotype of rs7038G/T polymorphism site of ARL6IP5 gene and IS.In the codominant model,the frequency of TT,TG,GG genotype at the rs7038G/T polymorphism site was 31.2%,58.3%,10.5%and 46.9%,46.9%,6.2%in the IS group and the healthy group,respectively.There was a significant statistical difference between genotype frequencies(heterogeneous model:P<0.0001,OR=0.54,95%CI=0.38-0.75;homozygous model:P=0.005,OR=0.40,95%CI=0.21-0.76).In the dominant model,the difference was statistically significant(P<0.0001,OR=0.52,95%CI=0.37-0.72).There was no statistical difference in recessive patterns(P=0.70,OR=0.57,95%CI=0.31-1.05).A similar result was obtained by multivariate regression analysis after adjusting for age and sex.The frequency of ARL6IP5 gene rs7038G/T polymorphism in PSCI group and PSNCI group was 40.16%and 39.27%,respectively.There was no significant difference in the frequency of G allele of rs7038G/T polymorphism between PSCI group and PSNCI group(P=0.83,OR=0.96,95%CI=0.72-1.28).In co-dominant model,the frequency of TT,TG,GG genotype of rs7038G/T polymorphism in PSCI group and PSNCI group was31.31%,57.1%,11.6%and 30.9%,59.7%,9.4%,and that in PSNCI group was 31.31%,57.1%,11.6%and 30.9%,9.4%,respectively.There was no statistical difference between genotype frequencies(heterogeneous model:P=0.80,OR=1.06,95%CI=0.68-1.65;homozygous model:P=0.59,OR=0.82,95%CI=0.4-1.68).In the dominant model,compared with the TT genotype,the TG/GG genotype had nothing to do with the pathogenesis of PSCI(P=0.93,OR=1.02,95%CI=0.66-1.57).In the recessive model,compared with the TG/GG genotype,the GG genotype was not associated with the pathogenesis of PSCI(P=0.48,OR=0.79,95%CI=0.41-1.52).After adjusting for age and sex,similar results were obtained by multi-factor Logistic regression analysis.Stratified analysis showed that there was no statistical difference in demographic characteristics and some clinical data of IS group in heterozygote model(P>0.05).In homozygote model,triglyceride analysis was statistically significant(P<0.01),but there was no statistical difference in other stratification analysis(P>0.05).The demographic characteristics and some clinical data of PSCI group in heterozygote mode had no statistical difference in PSCI group(P>0.05),but there was statistical significance in PSCI group in glycerol stratification analysis under homozygote mode(P<0.01).There was statistically significant difference in sex stratification(P<0.009),but there was no statistical difference in other stratification analysis(P>0.05).(4)TT,GG,TG,TG/GG of ARL6IP5 gene rs7038G/T polymorphism was associated with the polymorphism of ARL6IP5 gene,but no significant difference between TG and TG/GG genotype and ARL6IP5 gene mRNA and protein in healthy group(P>0.05)found,similar results were obtained in the IS group,PSCI group and PSNCI group,according to the relationship between the genotype of ARL6IP5 gene rs7038G/T polymorphism and its mRNA and protein.Conclusions:(1)Multivariate analysis showed that the percentages of neutrophils,platelet,triglyceride and creatinine were correlated with the incidence of IS.And the degree of education,ADL and history of heart disease might be associated with the pathogenesis of PSCI.(2)ARL6IP5 gene mRNA and protein may be involved in the pathophysiological process of IS and PSCI.(3)The polymorphism of ARL6IP5 gene rs7038G/T may be associated with the genetic susceptibility of IS.G allele and GG genotype may be protective factors of IS,which may be associated with triglyceride,but not affected by age,sex or percentage of neutrophils,high-sensitivity C-reactive protein,uric acid and other factors;rs7038G/T polymorphism may not be associated with genetic susceptibility to PSCI.(4)The transcription and translation levels of ARL6IP5 gene was not changed caused by rs7038G/T polymorphism genotype. |
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