调控miRNA成熟通路中SNPs与新疆维吾尔族结核病遗传易感性及后续功能分析

The prevalence of tuberculosis is high in Xinjiang and mortality is higher than the national average level. Prophase research of our group shows that the higher prevalence of tuberculosis is related to the unique host genetic susceptibility. Research shows that the genetic variation(single nucleotide polymorphism, SNP) of each component of micro RNA(miRNA) mature path may lead to abnormal miRNA formation, and at last become disease risk factors through the miRNA silence effect. This topic will study the relations between susceptibility to tuberculosis and miRNA of molecular genetic correlation in Uygur of Xinjiang. Characteristic candidate gene in miRNA mature path of patients with tuberculosis in Uygur will be selected using bioinformatics methods, and high resolution melting(HRM) method will be used to detect allele and genotype frequency distribution of SNPs selected from all patients and control samples. Discrepant SNPs of allele frequency distribution between patients group and control group will be analyzed through the SNPs genotyping comparison with control group and case-control study, and unbalanced transmission of SNPs will be detected to analyze the relevance between SNPs and patients with tuberculosis. By the luciferase recombination carrier method, Real-time RT-PCR and bioinformatics method, the SNPs will be confirmed and their functions will be analyzed to identify the role of SNPs in occurrence and development of tuberculosis, Through this, we can investigate the SNPs genetic diversity in gene sequence of miRNA maturation path related to regulation of tuberculosis infection in Uygur, and know the molecular mechanisms of miRNA expression and regulation.

课题组前期研究发现结核病的发生发展与不同人群独特的自身遗传背景密切相关有关。调控miRNA成熟通路中各成分的遗传变异（SNPs）可能会导致miRNA生成异常，进而影响靶基因转录后调控，从而成为疾病发生的危险因素。本课题拟利用生物信息学方法筛选新疆维吾尔族患者miRNA成熟通路中特有的侯选基因，采用HRM方法对筛选获得的SNPs在结核病患者/对照人群中等位基因及基因型频率的分布进行分型，通过和对照人群的SNPs基因分型比较及病例对照研究，分析患者组和对照组等位基因与基因型频率分布有差异的SNPs，分析SNPs 与新疆维吾尔族结核病人的关联性。通过荧光素酶重组载体方法、Real time -PCR 以及生物信息学技术进行SNPs的确证及后续功能分析，明确这些SNPs 在新疆维吾尔族结核发病过程中的分子信号通路与相关miRNA的作用机制，为最终阐明遗传变异影响新疆维族结核发病的分子机理奠定基础。

本项目围绕miRNA成熟通路上的SNPs是否影响新疆维吾尔族人群结核病的患病危险及影响的分子机制进行探讨。首先，通过对新疆维吾尔族结核病患者血清差异表达miRNA筛选及功能分析，验证了新疆维吾尔族结核病患者血清中存在差异表达的miRNA，并通过生物信息学软件和通路分析预测差异miRNA通过其靶基因参与多个生物学过程和信号通路调节，与结核病感染和宿主免疫调控有关。进而利用生物信息学筛选了miRNA 成熟通路中9个基因的20个SNP位点，通过对311例维吾尔族健康人和465例维吾尔族结核病患者DNA样本相关SNP位点进行分型。然后通过病例对照研究，分析新疆维吾尔族结核病患者与上述SNP的相关性。结果显示4个阳性的SNP位点与新疆维吾尔族结核病遗传易感性具有相关性。我们进一步对4个阳性位点所在基因mRNA表达量进行qPCR表达定量和ELISA检测，探讨调控miRNA 成熟通路中SNPs 是否是通过影响本身基因的表达和蛋白量的表达影响到下游microRNA成熟。结果表明调控miRNA 成熟通路中SNPs可能通过影响其所在基因的表达量，影响下游microRNA成熟在新疆维吾尔族结核病发生发展过程中发挥作用。综上所述，本研究从 miRNA 为核心的基因网络角度为新疆维吾尔族人群结核发病相关的分子机理提供了进一步探讨的基础。