miRNAs介导CD147基因3’-UTR多态性对缺血性脑卒中的关联研究

Cerebral ischemic stroke is a complicated multifactor disease caused by environmental and hereditary factors. The genetic susceptible candidate genes are still unclear. CD147 is involved in the pathophysiological process of cerebral ischemic stroke, which suggests that CD147 gene is one of the possible candidate genes for cerebral ischemic stroke. The study is performed to detect CD147 mRNA and protein in PBMCs of the cerebral ischemic stroke patients and healthy individuals by Real-time PCR and Western blotting; and to analyze the association between CD147 3’-UTR polymorphisms (rs8259, rs6758, rs13676 and rs1063376) and cerebral ischemic stroke genetic susceptibility, and also the relation with CD147 mRNA and protein in PBMCs using RFLP-PCR.The above mentioned polymorphisms might influence the identification and binding capacity of miRNA-492, miRNA-491, miRNA-485 and miRNA-146a respectively, luciferase reporter assay will be used to determine the effect of the SNPs on CD147; furthermore, Real-time PCR and Western blotting will be used to validate the CD147 mRNA stability and translation efficiency after miRNAs mimics and inhibitors transfection in order to appreciate the biologic effect of miRNAs binding sites SNPs in CD147 3’-UTR in cerebral ischemic stroke.

缺血性脑卒中是环境和遗传因素所致多因素疾病，其遗传易感基因并不清楚。CD147参与缺血性脑卒中病理生理过程，可能是其遗传易感候选基因之一。本项目拟采用实时荧光定量PCR和免疫印迹检测CD147 mRNA和蛋白在缺血性脑卒中外周单个核细胞的表达水平；RFLP-PCR分析CD147 3’-UTR多态性(rs8259、rs6758、rs13676和rs1063376)与缺血性脑卒中的遗传易感性、及与外周单个核细胞CD147 mRNA和蛋白的关系。上述多态性可能分别影响miRNA-492、miRNA-491、miRNA-485和miRNA-146a的识别、结合能力，用荧光素酶报告基因分析上述多态性对CD147的影响；实时荧光定量PCR和免疫印迹分析外源miRNAs模拟物和抑制物对CD147 mRNA稳定性及翻译水平的影响；阐明CD147 3’-UTR 多态性在缺血性脑卒中的生物学效应。

Real-time PCR 和Western blot检测缺血性脑卒中患者和健康人群PBMCs 中CD147 mRNA 和蛋白表达；RFLP-PCR检测CD147 3’-UTR 多态性(rs8259 A/T、rs6758 A/G、rs13676 A/G、rs1063376 C/T等)分布情况，分析基因型对CD147 mRNA 和蛋白的影响。上述多态性可能分别影响miRNAs识别结合能力，荧光素酶报告基因检测其与CD147稳定性；Real-time PCR和Western blot分析miRNAs模拟物和抑制物对CD147 mRNA和蛋白的变化。.CD147 mRNA在缺血性卒中组表达明显降低，CD147蛋白在缺血性脑卒中无显著变化。CD147 rs8259 A/T多态性A等位基因和携带A基因型是缺血性卒中的风险因素，rs1063376 C/T多态性C等位基因和携带C基因型是其保护因素。rs6758 A/G和rs13676 A/G多态性与缺血性卒中没有相关性。rs8259 A/T基因型AA和AT 使其 mRNA水平明显升高。rs1063376 C/T基因型不影响CD147 mRNA和蛋白。筛选出rs8259 A/T和rs1063376 C/T可能受miRNAs调控。共转染pcDNA3.1-miR-492和pGL3 rs8259-AA或pGL3 rs8259-TT到HUVCs，AA基因型（野生型）荧光素相对活性较TT基因型（突变型）减少约30%，野生型和突变型细胞株共转染pcDNA3.1-miR-492其相对荧光素酶活性显著抑制。CD147 mRNA水平在野生型明显升高，突变型加入miR-492模拟物其mRNA表达明显降低，miR-492直接与rs8259 TT结合从而降低其 mRNA表达水平。共转染pGL3-rs1063376-CC（野生型）、pGL3- rs1063376-TT（突变型）和pcDNA3.1-miR-146a，其荧光素相对活性结果与rs8259A/T类似，但miR-146a不影响CD147 mRNA和蛋白表达。. CD147基因及其rs8259 A/T和rs1063376 C/T多态性与缺血性卒中遗传易感性有关。miR-492直接与rs8259 TT结合从而降低CD147 mRNA表达水平，miR-146a不影响CD147 mRNA和蛋白的表达。.