基于miRNAs介导的TLRs信号传导网络研究肾阳虚证免疫稳态失衡及右归丸的修复作用机制

Our prophase work indicates that immune homeostasis imbalance is one of the basic pathogenesis of Kidney-Yang Deficiency Syndrome. The one of evidences of this conclusion is that some genes in downstream of Toll-like receptors (TLRs) signal pathway were down-regulated in patients with Kidney-yang Deficiency Syndrome, which suggests that these downstream genes in TLRs signal pathway were negatively regulated in Kidney-yang Deficiency Syndrome. Considering the key action of negative regulation of gene expression for miRNA, we infer that miRNA maybe is the important regulatory element in immune homeostasis imbalance of Kidney-Yang Deficiency Syndrome. To confirm this inference, we plan to design this project to identify the networking regulated pattern of miRNAs on TLRs signal transduction network for Kidney-Yang Deficiency Syndrome. The miRNAomics and bioinformatics approaches will be applied. The Kidney-Yang Deficiency case group, healthy control group and Youguiwan treatment group (each group includes 20 subjects) will be selected to perform miRNA microarray and TLRs Signaling Pathway PCR Array experiments. The differently expressed miRNAs and TLRs signal pathway genes will be screened between case group and control group, between before and after Youguiwan treatment. The overall research will be performed at four steps: First, screening the differently expressed miRNAs and their target genes; Second, quantitating the expression levels of genes of TLRs signal pathway; Third, performing Correlation Analysis between target genes of screened miRNAs and genes in TLRs signal pathway, and constructing the miRNAs mediated TLRs signal transduction network; Fourth, positioning the core miRNA-TLR couples in the constructed network. The repairing molecular pathway of Youguiwan on Kidney-yang deficiency Syndrome will be also identified. This study plans to integrate multidisciplinary study strategy such as miRNAomics approaches to the complicated pathogenesis of Kidney-Yang Deficiency Syndrome. By detecting the relationships between certain miRNAs and immune genes, we try to explore the molecular foundation of immune homeostasis imbalance and repairing mechanism of Youguiwan on Kidney-Yang Deficiency Syndrome at post-transcriptional level. As a result, the study may provide scientific basis of pathogenesis for Kidney-yang Deficiency Syndrome, and thus help us to formulate corresponding prevention or treatment measures for the disease.

肾阳虚证病机特征之一为免疫稳态失衡，其佐证之一为发现肾阳虚证患者经典的免疫通路- - TLRs信号通路下游基因显著下调，提示该通路基因表达存在负调控。联系miRNA负调控基因表达功能，我们推论，miRNA可能是肾阳虚证免疫稳态失衡的重要调控元件。 为此，本计划拟采用miRNA组学及分子网络建模方法，筛选肾阳虚证病例组、健康对照组及右归丸治疗组各20例，组间及治疗前后对照作如下研究：①差异表达miRNAs及其靶基因筛选；②TLRs信号通路基因表达定量；③数据融合，差异miRNAs靶基因与TLRs通路基因关联分析及调控网络建模；④定位核心miRNA-TLR调控对并对照分析右归丸修复作用路径。 本项目整合miRNA组学等多学科结合策略，将证候病机复杂特性和miRNA调控相对接，从免疫基因表达的转录后调控水平探索肾阳虚证免疫稳态失衡分子架构及右归丸修复作用机制，为肾阳虚证病机理论及其防治提供科学依据。

本项目筛选健康对照组（10例）及肾阳虚证（20例），开展了以下四个方面的研究：1.肾阳虚证患者右归丸疗效分析：在200例被调查的患者中，纳入肾阳虚证患者50例，完成了2个月的右归丸疗效观察。筛选健康对照10例及治疗显效者20例，分别于右归丸治疗前后完成理化检测和标本采集。2.差异表达miRNAs及其靶基因筛选：分别采集3组受试者外周血5mL，提取白细胞，完成RNA抽提及质检，应用miRCURYTM LNA Array芯片技术筛选3组间差异miRNAs。结果共筛选到肾阳虚证相关差异miRNAs 48个，右归丸疗效相关差异miRNAs 435个，其中有16个miRNAs经右归丸治疗后其表达水平出现显著变化，并且其中12个miRNAs的表达回归至健康对照组水平（P>0.05）。3. 差异表达TLRs信号通路基因mRNA定量：分别取制备的3组RNA样品，与人TLRs信号通路PCR芯片杂交，获取各组TLRs信号通路基因的定量表达数据， 2-ΔΔCt法分析各组基因的表达差异。结果得到与肾阳虚证相关差异基因28个，与右归丸疗效相关差异表达39个，其中有12个基因经右归丸治疗后其表达水平出现显著变化，且其中 9个基因的表达回归至健康对照组水平（P>0.05）。4. 差异表达miRNA对TLRs信号通路基因的网络调控：利用TargetScan 和 miRbase数据库对16个差异miRNA进行靶基因预测，以catescape数据库构建差异miRNA及其靶基因的调控网络，根据miRNAs与TLRs信号通路基因之间的调控关系，定位出25个核心miRNA-TLRs信号通路基因调控对，其中20个呈反向调控关系。本项目从免疫基因表达的转录后调控水平探索肾阳虚证免疫稳态失衡分子架构及右归丸修复作用机制，为肾阳虚证病机理论及其防治提供科学依据。共发表论文14篇，获得厅局级科技奖1项，培养硕士研究生5名。