m6A去甲基化酶ALKBH5和修饰识别蛋白YTHDF3介导的lncRNA-GAS5降解途径调控YAP靶基因参与糖尿病肾脏损伤

在前期研究发现糖尿病肾病患者血清和肾脏lncRNA GAS5表达下调参与肾脏损伤，高糖培养的肾小管上皮细胞中GAS5的m6A水平增加，去甲基化酶ALKBH5和修饰识别蛋白YTHDF3表达变化的基础上，从人群、动物、细胞三个层面分析DN发生发展过程中RNA m6A修饰的生物学规律和作用。利用CRISP-Cas9、腺相关病毒、慢病毒和小分子抑制剂靶向调控肾脏ALKBH5、YTHDF3、GAS5和YAP的表达，验证长链非编码RNA的m6A修饰机制，探讨ALKBH5/lncRNA GAS5/YAP/YTHDF3负反馈调节环路对糖尿病肾病关键致病因子YAP靶基因desmin、snail2、CyclinD1、CTGF、AREG等的转录，对足细胞和肾小管上皮细胞失分化、细胞骨架重排、EMT、凋亡、足细胞脱落，纤维化的影响，旨在从表观遗传学和转录后修饰角度阐明DN的发病机制，为DN的诊治提供新思路。

In our previous study, we found that the serum and renal lncRNA GAS5 levels in patients with diabetic nephropathy (DN) were down-regulated and involved in kidney injury. The m6A level of GAS5 was increased, and the expression of demethylase (eraser) ALKBH5 was decreased, and the expression of m6A reader YTHDF3 was increased in renal tubular epithelial cells treated with high glucose. In this study, we will do clinical, in vivo and in vitro experiment to analyze the dynamic changes and the pathogenic role of RNA m6A methylation modification during the development of diabetic nephropathy. CRISP-Cas9, adeno-associated virus, lentivirus and small molecule inhibitors will be used to regulate the renal expression of ALKBH5, YTHDF3, GAS5 and YAP to verify the m6A methylation modification mechanism of long non-coding RNA, and to investigate the effect of ALKBH5/lncRNA GAS5/YAP/YTHDF3 negative feedback loop on the YAP target genes including desmin, snail2, CyclinD1, CTGF and AREG, which are the key pathogenic factors of diabetic nephropathy involved in podocyte and renal tubular epithelial cell dedifferentiation, cytoskeleton rearrangement, podocyte aberrant mitosis, epithelial-mesenchymal transdifferentiation, apoptosis, podocyte detachment and fibrosis. It aims to clarify the pathogenesis of diabetic nephropathy from a new perspective of epigenetics and post-transcriptional modification mechanism and to identify novel diagnosis and treatment target.