靶向抑制肾脏microRNA-503和microRNA-181d表达治疗KKAy小鼠糖尿病肾病的机制研究

Restoring or inhibition expression of miRNAs provide a new approach to management of diabetic nephropathy(DN). Our previous proteomics and microarray studies suggested that the expression of miRNA-503 and miRNA-181d were significantly up-regulated in the glomeruli of KKAy mice and inhibited by losartan treatment. In present study, miRNA-503 knockout or miRNA-181d knockout plasmids will be delivered into the living kidney of diabetic KKAy mice by ultrasound-microbubble-mediated gene transfer, and the renoprotective role will be observed. By in vitro study, glomerular mesangial cells, endothelial cells and podocyte will be transfected by miRNA-503 and miRNA-181d mimic or knockout plasmids respectively. Apoptosis,injury and proliferation assay of renal intrinsic cells cultured under high glucose will be conducted. The effect of miRNA-503 and miRNA181d on protein expression profiles of KKAy mice glomeruli isolated by Dynabeads and intrinsic cells under high glucose will be investigated using 2D-DIGE and MALDI-TOF mass spectrometry.The expression and function of predicted target genes of miRNA-503 and microRNA-181d including GRP75, Wnt3a, PI3K, AKT and BCL-2 will be observed. The objective is to identify the regulatory networks and the crosstalk of miRNA-503 and miRNA-181d and to explore novel therapeutic targets for DN.

miRNA功能的调控为糖尿病肾病（DN）治疗提供了新靶点。本研究拟在前期2型糖尿病KKAy小鼠肾小球蛋白和miRNA表达谱研究的基础上，选择在KKAy小鼠肾小球内表达明显上调且其异常表达可被氯沙坦抑制的miRNA-503和miRNA-181d，应用超声微泡技术，分别向KKAy小鼠肾脏转染miRNA-503和miRNA-181d的敲除质粒，观察其对DN的治疗作用；体外培养肾小球系膜细胞、内皮细胞和足细胞，分别转染miRNA-503和miRNA181d的过表达和敲除质粒，观察其对高糖培养的3种肾脏固有细胞损伤、凋亡、增殖能力的影响。应用蛋白组学技术，检测转染后小鼠磁珠分离肾小球和3种固有细胞蛋白表达谱的变化，进行GRP75、Wnt3a、PI3K-AKT、BCL-2等关键靶基因的功能分析。旨在明确miRNA-503和miRNA-181d在DN发病中的网络式调控作用，为DN的诊治提供新思路和新靶点

本研究在2型糖尿病db/db小鼠和高糖培养的足细胞、系膜细胞和肾小管上皮细胞中探讨miR-503、miR-181d、miR-148b和miR-21调控网络在糖尿病肾脏损伤机制中的作用及干预效果。分析糖尿病和糖尿病肾病患者循环miRNA和lncRNA表达谱的变化，筛选与DN发生密切相关的miRNAs和lncRNAs，寻找新的发病机制和生物标志物。并探讨高糖代谢记忆是否通过表观遗传学机制介导糖尿病肾脏损伤。结果发现2型糖尿病db/db小鼠肾组织中miR-503、miR-21、lncRNA-ARAP1-AS1/AS2及其靶基因ARAP1存在差异表达，通过腺相关病毒质粒靶向抑制2型糖尿病db/db小鼠肾组织miR-503过表达可以减轻尿白蛋白排泄率，系膜基质增生和足细胞损伤。体内和体外的研究证明miR-503可通过调控靶基因CCNE1和Bcl-2的表达，促进系膜细胞G2期向S期转换，促进细胞增殖和细胞外基质蛋白分泌，活化caspase酶，诱导线粒体途径凋亡，抑制miR-503表达可减轻肾脏损伤。抑制miR-148b的表达，可上调AMPKα1的表达，抑制内质网应激亢进，减少细胞外基质蛋白的堆积；miR-21可通过调控PTEN/Akt/mTOR信号通路，促进系膜细胞肥大、增殖、抑制系膜细胞和足细胞自噬，miR-21抑制质粒能够缓解系膜细胞和足细胞自噬抑制，减轻糖尿病肾脏损伤。乌索酸通过抑制Wnt/β-catenin和miR-21靶向的PTEN/Akt/mTOR信号通路活性，恢复高糖诱导的自噬抑制，降低db/db小鼠体重、血糖和尿白蛋白排泄率，抑制足细胞上皮间充质转分化，缓解系膜基质增生和足细胞损伤。临床研究发现，在糖尿病和糖尿病肾脏损伤的发生过程中，循环miR-1179的表达逐渐上调，miR-148b、miR-150的表达逐渐下调，miR-21的表达上调，lncRNA-GAS5的表达下调，表达下调的lncRNA-ARAP1-AS1和表达上调的lncRNA-ARAP1-AS2作用于靶基因ARAP1，参与了糖尿病和糖尿病肾病的发生，有望成为新的生物标志物。短暂高糖培养可诱导系膜细胞组蛋白甲基化转移酶set7/9和组蛋白H3K4单甲基化的表达上调，促进肾小球系膜细胞NF-κB p65，炎症因子MCP-1、VCAM-1的持续高表达。