预测miRNA在原发性高血压合并2型糖尿病的治疗靶点研究

。Essential hypertension(EH) complicated with type 2 diabetes mellitus（T2DM） is a kind of common polygenic inherited disease, which may induce cardio-cerebrovascular disease.Carrying out the research on the gene regulatory mechanism of the insulin resistance significantly associated with EH complicated with T2DM is important to clarify the genetic pathogenesis of the disease, the risk assessment, the evaluation of the prognosis, the prevention of the disease and screen the therapeutic target of genes. Based on the research of the association of AGTR1,CMA1 gene polymorphisms with essential hypertension in populations of Yunnan province, we hypothesis that microRNAs are involved in the regulation of target gene expression related to the occurrence of the EH Complicated with T2DM. Finally, we could verify the hypothesis we proposed before, explore the signal pathway of microRNA regulatory mechanism of the insulin resistance, reveal the molecular mechanism of the occurrence of the EH complicated with T2DM in populations of Yunnan province, and predict the therapeutic target of microRNA by researching on target gene expression regulated by microRNA. The differential expression of microRNA can be used as one of the diagnostic criteria of EH complicated with T2DM. At present, we have not seen the relevant report at home and abroad. This study for the first time reveals that microRNA regulatory mechanism of target gene expression is likely to be the molecular mechanism of the occurrence of the EH complicated with T2DM. It can provide theoretical basis for the prevention and treatment of EH complicated with T2DM, and put forward new ideas for the application in clinical treatment.

原发性高血压（EH）合并2型糖尿病（T2DM）是并发心脑血管病多、治疗难度大的常见多基因遗传疾病，开展与EH合并T2DM发病密切相关的胰岛素抵抗调控基因研究，对阐明其遗传发病机制、风险评估、愈后判断、预防及基因治疗靶点筛选均具有重要意义。我们近年在对AGTR1、CMA1等基因多态与云南人群EH发病相关的研究基础上，提出miRNA参与EH合并T2DM及并发症发生靶基因表达调控的假说。通过研究miRNA调控靶基因的表达来验证假说，探索与胰岛素抵抗作用相关miRNA信号通路，揭示云南汉族EH合并T2DM发病的分子机制，预测miRNA治疗该疾病的作用靶点； miRNA差异表达可能作为EH合并T2DM及并发症发生判定参考指标之一，此法目前国内外文献未见报道，本研究首次揭示miRNA调控靶基因表达可能是EH合并T2DM及并发症发生的分子机制，为其防治提供理论依据，并为应用于临床治疗提供新的思路。

原发性高血压（Essential hypertension EH）常合并2型糖尿病（Type 2 diabetes mellitus T2DM）,二者并存时，内皮细胞和血管功能受损更加严重，心脑血管疾病的发病率高达50％，EH 合并 T2DM 发病率高、并发心脑血管病多、治疗难度大，目前对其发病机制不清楚，针对性的有效治疗和预防具有十分重要的临床意义。本研究通过检测 EH 、T2DM、EH合并T2DM和 NC组血浆 miRNA，筛选出差异表达miR-197、miR-130a、miR-195和miR-27a，进行qPCR验证，与 NC组比较，发现miR-197、miR-130a、miR-195和miR-27a在EH 、T2DM、EH+T2DM组表达下降，在EH合并T2DM组中下降尤明显。应用生物信息学技术TargetScan、 miRanda和DIANA-micro程序预测miR-195作用的靶基因是DRD1和 LDLRAD2。通过荧光报告基因分析验证了DRD1和 LDLRAD2是miR-195的一个直接的靶标。过表达miR-195可以显著的抑制DRD1和 LDLRAD2 3’UTR的荧光活性，当DRD1和LDLRAD2 3’UTR与miR-195的结合位点的种子序列突变之后，这种抑制作用消失。通过PCR方法，扩增并测序靶基因DRD1的3’UTR区，证实DRD1是miR-195的靶向基因，检测靶基因DRD1的3’UTR区（ miRNA靶结合区域）的SNPS位点，提示DRD1 3’UTR的SNPS检测发现一些碱基突变，在一定程度上，说明遗传多态与EH 合并T2DM的易感性有着一定的关联，也为揭示EH 合并T2DM 及并发症发生的分子机制奠定一定的基础，因此，DRD1可能成为治疗EH 合并T2DM的潜在的治疗新靶点。