基于系统生物学与中医整体观探索miRNA-mRNA调控网络影响缺血性中风及其证候的机制

MiRNA is a very important biological marker of ischemic stroke (IS). However, research on the regulation mechanism of miRNA on the Traditional Chinese Medicine (TCM) syndromes of IS has not been reported. Based on the systems biology approach, we have previously constructed an IS-related miRNA-mRNA regulatory network. Our hypothesis is that the polymorphic regulation mechanism induced by this miRNA network has an effect on the development of IS and its TCM syndromes. Firstly, we use the Sequenom technology to perform the genotyping on SNPs located in the 3'UTR region of the target genes within the network, in order to explore the effect of the risk loci in the miRNA binding region on the development of IS and its TCM syndromes in the population level. And then we demonstrate how the polymorphic site in the 3'UTR region affect the binding of miRNA with the target genes, and the influence of miRNA in the stability and transcription of the the target genes, through laboratory technique, such as in vitro cell experiment, dual-luciferase reporter gene system, RT-PCR, Western blot etc., thus to illustrate the mechanism on the influence of miRNA-mRNA regulatory network in the risk of IS and its TCM syndromes. Our findings may provide a molecular biological basis for the ischemic stroke TCM syndrome differentiation and molecular target for treatment of stroke, and promote the TCM syndrome differentiation and personalized medicine.

miRNA是缺血性脑卒中(IS)的重要生物标志，但有关缺血性中风中医证候miRNA调控机制的研究尚未见报道。我们前期基于系统生物学方法构建了IS相关miRNA-mRNA调控网络，并假设该网络通过miRNA介导的多态调控机制影响IS及其中医证候的发生发展。本研究拟首先运用Sequenom对网络中靶基因3'UTR区功能位点的基因分型，从人群水平探明影响缺血性中风及其证候发生的miRNA结合区风险位点；然后，通过体外细胞实验，结合双荧光素酶报告基因系统、RT-PCR、Western blot等实验技术，研究靶基因3'UTR区风险位点对miRNA与靶基因识别结合的影响、以及miRNA对靶基因稳定性及翻译的影响。从整体上阐明 miRNA-mRNA调控网络影响缺血性中风及其中医证候发生发展的机制。研究结果可为缺血性中风中医辨证提供分子生物学依据，为中风治疗提供分子靶点，促进中医辨证论治和个体化医疗。

本项目首先在774例缺血性中风（IS）病例、793例对照样本中，利用Sequenom MassARRAY基因分型技术，对60个单核苷酸多态性（SNPs）进行基因分型检测。通过大样本人群关联研究，发现CRNKL1 基因rs16981448、C14orf119 基因rs6736、SNAPC3 基因rs4741506多态性与缺血性脑卒中易感性显著关联。本项目还鉴别出与缺血性中风不同中医证候发生风险显著关联的基因位点：3个SNPs(WDR5B基因rs1067、EGR3 基因rs11136094、SNAPC3基因rs12093)与IS风证发生风险显著关联；3个SNPs(WDR5B基因rs9843165、TBX21基因rs11657479、ZNF304基因rs7250521)与IS火热证发生风险显著关联；4个SNPs(CRNKL1基因rs12624987、ABLIM3基因rs9512、SCRN1基因rs11540586、ZNF260基因rs34550124)与IS痰证发生风险显著关联；2个SNPs(SH3BP5-AS1基因rs11713836、rs2046725)与IS血瘀证发生风险显著关联；4个SNPs(SERPINB9P1基因rs375556、TMEM63C基因rs383459、rs384491、rs3813539)与IS气虚证发生风险显著关联，上述16个SNPs与IS其它中医证候无显著关联。进一步双荧光素酶报告基因实验发现，C14orf119基因rs6736、SCRN1基因rs11540586位点可通过影响miRNA介导的结合抑制作用从而影响基因表达。本研究鉴别出了影响缺血性中风易感性、以及IS不同中医证候发生风险的基因风险位点，初步阐明了miRNA介导的多态性调控机制对缺血性中风及其中医证候发生机制的影响。研究结果为中风发生机制提供新的遗传学依据，为“同病异证”中医理论提供了有力支持，为精准中医辨证提供了分子遗传学依据，对促进中风二级预防、丰富中医基础理论、推动中医药发展有积极的意义。本课题已发表SCI论文4篇、中文论文7篇；培养硕士研究生5名、博士研究生1名，其中2人已获得硕士学位、4人在读。

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