| Background:Ischemic stroke (IS) is the most common type of stroke in clinic. The etiology of IS includes narrow or occlusion of cerebral vessels caused by atherosclerosis (AS), cerebral lacuna infarction, and cerebral thrombosis. Hypertension, AS, and increase in number of circulation platelet contribute greatly to cerebral infarction and cerebral thrombus events. Epoxyeicosatrienoic acids (EETs) are a group of endogous vasoactive substances that are produced during arochiodonic acid metabolism mediated by cytochrome P450. EETs are previously reported to exert anti-inflammatory, vasodilatory, and anti-platelet activities, and can inhibit the development of hypertension and AS. Soluble epoxide hydrolase (sEH) is the enzyme involved in the inactivation of EETs. Genetic polymorphisms in the coding gene of sEH, namely EPHX2, have been reported to be associated the risk for some AS related diseases such as coronary heart disease. MicroRNA 146a (miR-146a) is one of the microRNAs (miRNAs) that play important role in the regulation of inflammation. Several immune and inflammation related cytokines such as lipopolysaccharide (LPS), interleukin 1 (IL-1) and tumour necrosis factor (TNF-α) can stimulate the expression of miR-146a expression. A functional rs2910164 (G/C) polymorphism that occurs in the pre-miR-146a has been reported to affect the maturation of miR-146a. Our previous bioinformatics analysis of the 3'-untranslated region (3'-UTR) of human EPHX2 have found that the two single nucleotide polymorphisms (SNP) rs 1042032 A/G and rs 1042064 T/C are in complete disequilibrium, and both are located in potential miRNA binding site(miR-183 for rs1042032 and miR-576-3p for rs1042064). Furthermore, a potential miR-146a binding site was also observed in the EPHX2 3'-UTR. We hypothesize that both miR-146a and EPHX2 are candidate susceptibility genes of IS, and there might be interactions between genetic polymorphisms in these genes in affecting the risk of IS.Objective:To explore the associations between miRNA-146a rs2910164 and EPHX2 rs 1042032 polymorphisms with risk for IS in Changsha Han population by case-control study.Methods:Blood samples from IS patients and age- and sex-matched healthy controls were collected and DNA samples were extracted. The miR-146a rs2910164 (G/C) and EPHX2 rs 1042032 A/G polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Real-time PCR was performed to determine mature miR-146a and EPHX2 mRNA expression levels in peripheral blood mononuclear cells (PBMCs) from IS cases and in HUVECs after pretreatment with miR-146a mimcs for 24 hours. Bi-luciferase reporter assay was used to analysis the function of EPHX2 rs1042032 A/G and rs1042064 C/T polymorphisms, as well as to assess whether EPHX2 is the target gene of miR-146a. Difference in genotype distribution between cases and controls were analyzed with 2 test. Associations of miR-146a and EPHX2 genetic polymorphisms with IS risk were analyzed by unconditional logistic regression.Results:Frequencies for EPHX2 rs1042032 AA, AG and GG were 35.7%,52.0% and 12.3%, respectively, in controls and 39.6%,46.2% and 14.2%, respectively, in IS cases. No significant difference in the genotype distribution of the rs1042032 polymorphism was observed between overall cases and controls (P>0.05). When substrated by classification of IS, the frequence for the rs1042032 AA genotype was significantly increased IS patients suffered from cerebral lacuna infarction than the controls (P=0.046). The frequence for the rs1042032 AA genotype also trended to be increased in IS patients suffered from larg vessele AS (P=0.058), but decreased significantly in patients by other causes (P=0.018). After correction for risk factors for IS by unconditional logistic regression, the rs1042032 AA was associated with marginically increased risk for cerebral lacuna infarction (OR= 1.428,95% CI: 1.000～2.089, P=0.051), and is associated with increased risk for IS caused related with large and small arterial vessels diseases (including atheroslerosic infarction and lacuna infarction) (OR=1.37,95%Cl: 1.039～1.793, P=0.023). When substrated by the occurrence of hyperlipidermia, genotype frequence for rs1042032 AA was significantly in IS patients than controls in individuals with hyperlipidemia (P=0.006). While in individuals with normal blood lipids, the genotype distribution of rs 1042032 was comparable between IS cases and controls. Frequencies for miR-146a rs2910164 CC, CG and GG genotypes were 35.1%,46.8% and 18.2%, respectively, in controls and 32.6%,48.5% and 18.9%, respectively, in IS cases. No significant difference in the genotype distribution of the rs2910164 polymorphism was observed between overall cases and controls. When substrated by classification of IS, the frequence for the rs2910164 CC genotype trended to be increased in AS related IS cases (P=0.091). Results from unconditional logistic regression analysis showed that the rs2910164 CC genotype was associated with marginally increased risk for AS related IS (OR=1.382,95%CI: 0.957～1.995,P=0.084). When stratified by the hyperlipidemia status, frequence for the rs2910164 CC genotype was significantly higher in cases than controls in individuals with normal blood lipids (P=0.038) but comparable in subjects with hyperlipidemia. When combine genotype of EPHX2 rs 1042032 and miR-146a rs2910164 was analyzed, concomitant carriers of the rs1042032 AA and rs2910164 CC genotype showed significantly increased risk for AS related IS as compared with carriers of the non-risk genotypes for both polymorhisms (OR=1.462,95%CI: 1.113～1.921, P=0.006), while carriers of one risk genotype for either polymorphism showed no increase in IS susceptibility. EPHX2 mRNA exprsssion in PBMC from IS patients trended to be increased with the EPHX2 rs1042032 AA, but significant difference was not observed amont the rs1042032 genotypes (P>0.05). Levels of miR-146a and EPHX2 mRNA correlated negatively in PBMC from IS patients (Pearson's r=-0.363, P=0.020). The miR-146a level was significant increased in PBMC from patients with the miR-146a rs2910164 CC genotype as compared with those from the GC and GG genotype (P=0.039 and 0.006, respectively). Carriers of the combined genotype rs1042032 AA/rs2-910164 CC showed significantly decreased PBMC EPHX2 mRNA and increased PBMC miR-146a than any other combinations of genotypes (P<0.05 and P<0.01, respectively). Reporter gene activity for plasmid bearing the EPHX2 3'-UTR sequence with the rs1042032 A- rs1042064 T haplotype was decreased significantly as compared that plasmid bearing the rs1042032 G- rs1042064 C haplotype (P=0.022). Co-transfection of miR-146a mimic inhibited the reporter activity significantly in plasma bearing the rs1042032 G- rs1042064 C haplotype in a dose-dependent manner, but did not affect the reporter activity in plasma bearing the rs1042032 A-rs1042064 T haplotype. miR-146a did not affected EPHX2 mRNA expression in cultured HUVECs..Conclusion:1) EPHX2 rs1042032 AA genotype is associated with increased risk for arterial vessel related IS and IS in individuals with hyperlipidemia in Changsha Han population; 2) miR-146a rs2910164 CC genotype might be associated with increased risk for large arterial related IS in Changsha Han population; 3) There are interactions between EPHX2 rs 1042032 and miR-146a rs2910164 polymorphisms in affecting IS risk in Changsha Han population; 4) The expression of miR-146a correlates negatively with EPHX2 mRNA in IS patients; 5) miR-146a rs 1042032 CC genotype can decrease PBMCs miR-146a expression in IS patients, and the combination genotype of rs 1042032AA/rs 1042032 CC is associated with decreased EPHX2 mRNA expression and increased miR-146a expression in IS patients; 6) Either rs 1042032 or rs 1042064 or both SNPs in EPHX2 3'-UTR are functional; 7) EPHX2 is a new target for miR-146a, and inhibitory effects of miR-146a on EPHX2 is dependent on rs1042032/rs 1042064 polymorphisms.
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