| Background:Cardio-cerebrovascular diseases such as atherosclerosis (AS), coronary artery disease (CAD), essential hypertension (EH), heart failure (HF), and stroke have become a great threat to human health and major causes of death worldwide. Though the clinical manifestations are diversed, there are some common characteristics for these diseases. Recent studies have suggested important role of inflammation in the development of cardio-cerebrovascular diseases. Inflammation bridges hypertension and AS, two major basic diseases for cardio-cebrovascular diseases. MicroRNA 146a (miR-146a) is one of the miRs that have been implicated in inflammation. Lipopolysaccharide (LPS), interleukin 1 (IL-1) and tumour necrosis factor a (TNF-a) are reported to stimulate miR-146a expression in a nuclear factorκB (NF-κB) dependent pathway. A functional rs2910164 (G/C) polymorphism that decreases the expression level and process efficiency of maturation on the passenger strand of miR-146a has been identified. The polymorphism lead to the production of miR-146a*G and miR-146a*C. We hypothesize that miR-146a is a candidate susceptibility gene of cardio-cerebrovascular diseases.Objective:To explore the association of the miRNA-146a rs2910164 polymorphism with risk for cardio-cerebrovascular diseases including EH, CAD, HF, and ischemic stroke.Methods:Blood samples from patients with EH (n=173), CAD (415), HF (555), and ischemic stroke (268) were collected. Bloods samples from 1010 age-and gender-match healthy controls of EH were also collected. The miR-146a rs2910164 (G/C) polymorphism were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). MiR-146a precussor (pre-miR-146a) and mature-miR-146a in peripheral blood mononuclear cells (PBMCs) from CAD cases and controls were detected by real-time PCR. Plasma levels of IL-5 and TNF-αwere measured by method of ELISA. PBMCs from healthy volunteers were isolated and treated with TNF-α(10 ng/mL) or oxidized-low density lipoprotein (ox-LDL,100 mg/L) for 24 hours, and pre-miR-146a were then detected by real time PCR. Bi-luciferase reporter assay was used to the identification of miR-146a target gene NADPH oxidase 4 (NOX4). Difference in genotype distribution and allele frequencies between cases and controls were analyzed withχ2 test. Associations of miR-146a genetic polymorphism with risk for diseases were analyzed by unconditional logistic regression. Effect of miR-146a polymorphism on the prognosis of HF was evaluated by Kaplan-meier survival analysis.Results:Grequencies for miR-146a rs2910164 CC, GC and GG genotypes in overall controls were 34.1%,45.0% and 20.8%, respectively. No difference in either genotype distribution or allelic frequencies between the controls from Changsha and Hiakkou regions was observed (p=0.646). No difference in genotype distribution of rs2910164 polymorphism between EH or CAD cases and controls was observed (p>0.05, respectively). In individuals with normal plasma lipid profile, rs2910164 CC genotype trended to be increased in CAD cases than controls (39.1% vs 33.9%), though significant difference was not observed. Significant differences in genotype distribution of the rs2910164 G/C polymorphism between HF cases or ischemic stroke cases and controls were observed (p=0.00019 for HF; p=0.010 for ischemic stroke), and individuals with the GG genotype was ovrepresented in both cases. As compared with carriers of the rs2910164 C allele, individuals with the rs2910164 GG genotype showed increased risk for heart failure (OR=2.08,95%CI:1.474-2.941,p=0.000032) and ischemia stroke (OR=1.250,95%CI:1.067-1.463, p=0.006). Frequency of rs2910164 GG genotype in relapse and readministration HF cases 6 months within discharge trended to be increased, however, significant difference in incidences of relapse and readministration among rs2910164 genotypes was not observed (p>0.05). As compared with controls, the expression levels of pre-miR-146a and mature-miR-146a in CAD cases was decreased significantly (p=0.016 and 0.080, respectively). When stratified by clinical type of CAD, the decrease in pre-miR-146a and mature-miR-146a expression in PBMCs were obvious for patients with stable angina pectoris (p<0.05, respectively) but not for those with unstable angina pectoris, while miR-146a expression in PBMCs from patients with acute myocardial infarction trended to be increased as compared with the controls. Treatment of PBMCs with 10 ng/mL TNF-αincreased the expression of pre-miR-146a significantly (p<0.05), while treatment of PBMC with 100 mg/L ox-LDL decreased the expression of pre-miR-146a significantly (p<0.05). As compared with control subjects, plasma concentration of IL-5 was decreased significantly (p<0.05), while plasma concentration of TNF-αwas increased significantly (p<0.001). No difference in plasma concentration of IL-5 and TNF-αwas observed among miR-146a genotypes. However, when compared with controls with corresponding genotypes, significant decrease in plasma concentration of IL-5 and increase in TNF-a was observed only in CAD cases with the rs2910164 CC genotype. MiR-146a decreased the activity of pGL3-NOX4 in a dose-dependent manner, and significant inhibition was observed at miR-146a concentration of 1.0 nM (p<0.05).Conclusion:1) There is no difference in genotype distribution of the miR-146a rs2910164 in healthy Chinese controls from Changsha and Haikou regions; 2) MiR-146a rs2910164 polymorphism is not associated with risk of EH and CAD in Chinese;3) MiR-146a rs2910164 is associated with increased risk for HF and ischemic stroke in Chinese; 4) Rs2910164 GG genotype trends to be associated with increased incidence of CH relapse and readmission after discharge, confirmative conclusion await further study in larger popolation; 5) TNF-a and ox-LDL can increase and decrease the expression of PBMC pre-miR-146a respectively; 6) PBMC miR-146a expression is decreased in stable angina pectoris but trends to be increased in myocardial infarction; 7) NOX4 is a new target of miR-146a. |
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