miRNA调控血小板整合素αIIbβ3信号转导和骨架蛋白重构及其在冠心病血瘀证发病中的机制

Exploring novel antiplatelet agents has been the focus of coronary artery disease research. Our previous differential proteomics study showed that platelet integrin αIIb and gelsolin is correlated with coronary artery disease(CAD) with blood stasis syndrome(BSS). Latest research indicated that platelet microRNA(miRNA) suppresses the platelet protein expression. We speculated that integrin αIIbβ3 signaling and gelsolin remodeling played a key role in the pathogenesis of CAD with BSS, while the genes encoding these important proteins might be regulated by some special miRNAs. This project aimes to screen and identify the specific human platelet miRNAs of CAD with BSS by miRNA microarray and real-time PCR. Then the in-vitro human platelet activation and megakaryocyte differentiation model will be used. Based on target gene prediction,miRNA overexpression and silencing is to be performed to authenticate the correlation of these special miRNAs (such as let-7/hsa-miR-150, with integrin β3 as predicted target gene) levels with integrin αIIbβ3 signaling and gelsolin remodeling as well as platelet activation. It is of great significance for elucidating the melocular mechanism of CAD with BSS and providing new insights for the antiplatelet therapy by activating blood circulation herbs.

探寻更加安全高效的抗血小板药物一直是冠心病研究领域的焦点。课题组前期蛋白质组学研究表明血小板整合素αIIb及骨架蛋白Gelsolin与冠心病血瘀证相关。新近报道血小板内miRNA能抑制蛋白质表达，故推测血小板整合素αIIbβ3信号转导及骨架蛋白Gelsolin重构可能受某些miRNA的转录后调控，并在冠心病血瘀证发生中起重要作用。本项目拟采用miRNA芯片、荧光定量PCR等技术筛选和鉴定冠心病血瘀证特异性的血小板miRNA；并借助体外血小板活化和巨核细胞分化模型，结合靶基因预测、miRNA过表达和沉默等方法，研究let-7/hsa-miR-150(其预测靶基因为integrinβ3）等miRNA表达水平与整合素αIIbβ3信号通路、gelsolin重构及血小板活化的相关性。从miRNA靶向调控角度，为冠心病血瘀证分子机制及活血化瘀方药抗血小板治疗开辟新的研究途径，具有重要科学意义

血小板活化在冠心病血瘀证发生发展中发挥重要作用，血小板激活及继发血栓形成是导致急性冠脉事件的中心环节。血小板来源于骨髓巨核细胞，MiRNA参与血小板生成、mRNA调控、蛋白合成及血小板激活通路等。本项目采用高通量测序技术构建人血小板miRNA表达谱，筛选与冠心病血瘀证相关的血小板差异miRNAs；在靶基因预测的基础上，采用人巨核细胞诱导分化模型，通过miRNA过表达和沉默，及荧光素酶报告基因实验，验证miRNA对靶基因整合素β3和骨架蛋白gelsolin的调控；进一步观察活血化瘀中药对miRNA调控整合素β3和gelsolin的干预作用。本项目测序结果发现,人血小板表达的miRNA高达1021种，其中已知miRNA 887种, 未知miRNA 134种。通过KEGG数据库对差异表达miRNA进行靶基因预测，结果显示主要与血小板激活、血小板脱粒、血小板源生长因子受体信号通路、干细胞分化等有关。与整合素β3和gelsolin相关的冠心病血瘀证差异miRNAs有let-7a-3p 、miR-222-3p、miR-223-3p、let-7c-5p、miR-150-3p、miR-124-3p等。体外实验表明，AA、 F-actin刺激后人血小板CD62P活性升高，gelsolin含量明显增高。TPA诱导巨核细胞分化后let-7a 表达显著降低，miR-124表达显著升高；Integrin β3表达升高，Gelsolin表达降低。miRNA过表达或抑制实验、荧光素酶活性实验均证明let-7a可调控整合素Integrin β3表达，miR-124可调控骨架蛋白Gelsolin表达。与阿司匹林比较，活血化瘀中药三七总皂苷可使分化成熟的巨核细胞let-7a相对表达水平降低，Integrin β3水平升高；川芎嗪、三七总皂苷和阿司匹林三种药物均可上调miR-124表达水平，下调Gelsolin的mRNA和蛋白水平；miR-124 inhibitor可显著下调miR-124表达，继而抑制药物引起的Gelsolin下调。.本项目发现了在调控巨核细胞分化和血小板活化过程中Integrin β3和Gelsolin蛋白表达的miRNA，该成果可能为抗血小板药物治疗提供新的生物靶标和新的方法，具有重要科学意义和应用前景。