miR-124通过EGR1调控糖尿病肾病进展及肾脏纤维化的分子机制

Diabetic nephropathy (DN) is one of the most common causes of chronic kidney disease, leading to premature death and end-stage renal disease (ESRD). Inhibitors of the renin–angiotensin–aldosterone system (RAAS) have lowered the risk of albuminuria but cannot reduce the risk of ESRD. Thus, new therapeutic targets are urgently needed. Abundant experimental evidence indicates that the progression of DN are characterized by the activation of transforming growth factor beta (TGF-β), the accumulation of extracellular matrix (ECM) components in the glomeruli (glomerular fibrosis, glomerulosclerosis) and the tubular interstitium (tubulointerstitial fibrosis). Recently, early growth response protein 1 (EGR1) has been proved not only an upper signal molecule of TGF-β but also can activate the promoters of some components of ECMs. EGR1 may be a potential target for the prevention of the progress of DN. In our prior study, we found sitagliptin helped to resist the renal damage induced by high fat diet in apo E gene knockout mice (Apo E-/-) via potential inhibiting the expression of EGR1 gene. There are several microRNAs targeting EGR1. Especially, miR-124 has a negative correlation with the expression of EGR1 mRNA in mesangial cells incubated with high glucose. Thus, We hypothesis miR-124 targeting EGR1 may protect against the progression of kidney fibrosis in diabetic nephropathy. In the present study, the role of EGR1 will be first explored in the mesangial cells incubated with high glucose in vitro. The interaction between EGR1 and miR-124 will be analyzed by the methods of siRNA and RNA-ChIP. miR-124-mimic and LNA-inhibitor will be transfected into mesangial cells from wild type or EGR1-/- mice. Then the excretions and expressions of ECMs and the cell proliferation will be analyzed respectively. The RNA-ChIP will be employed to verify the direct relationship between the miR-124 and EGR1. At last, the mimic and LNA-miR-124 targeting EGR1 will be injected into DN mice models to understand the roles of miR-124 in the progression of DN. In the study, we will elucidate the protetive role of miR-124 targeting EGR1 in the progression of kidney fibrosis in DN.

我们在前期研究中发现，早期生长反应蛋白1（EGR1）可能作为miR-124的靶基因影响系膜细胞功能，抑制EGR1表达能显著改善高脂饮食诱导的apoE基因敲除小鼠的肾损害，推测miR-124可能通过抑制EGR1而防治糖尿病肾病（DN）的肾脏纤维化。本课题拟采用EGR1过表达和基因沉默的系膜细胞模型，探讨EGR1通过TGF-β发挥对DN进展及肾脏纤维化的重要调控作用；然后通过转染miR-124-mimic和LNA-miR-124观察miR-124对系膜细胞功能的影响，并以RNA-ChIP技术证实miR-124与EGR1之间的直接作用关系；最后以miR-124-mimic和LNA-miR-124腹腔内注射干预db/db的DN小鼠，观察DN进展情况。本课题将阐明miR-124抑制靶基因EGR1发挥对DN的肾脏保护作用，可能是防治DN肾脏纤维化潜在的新靶点和治疗途径。

糖尿病肾病（DN）是导致终末期肾病（ESRD）的主要原因之一，如何有效延缓或防止DN进展仍然是目前的研究热点和难点。我们在前期研究中发现，抑制肾皮质EGR1表达能改善高脂饮食诱导的apoE基因敲除小鼠的肾损害。体外实验显示EGR1表达在高糖刺激系膜细胞的早期呈显著升高，以EGR1为靶基因的microRNAs可能通过抑制EGR1及其下游信号通路发挥对DN的肾脏保护作用，可能是防治DN进展潜在的新靶点和治疗途径。本课题中我们发现自发性2型糖尿病大鼠与健康LETO大鼠相比，其肾脏EGR1和TGF-β1的mRNA及蛋白表达水平明显升高, 在体外培养的系膜细胞中，我们证实了高糖或TGF-β1刺激可快速上调Egr1表达，转染M61-Egr1质粒使系膜细胞高表达EGR1可显著增加系膜细胞层粘连蛋白（FN）、IV型胶原（ColIV）及TGF-β1表达，并促进系膜细胞增殖。相反，采用siRNA抑制EGR1表达则逆转上述情况。在体外培养的人近端肾小管上皮细胞株（HK-2），我们也证实了过表达EGR1促进TGF-β1和ECMs表达。免疫共沉淀(ChIP)分析结果提示Egr1可与TGF-β1启动子直接结合促进其转录表达。该部分结果提示EGR1通过直接作用于TGF-β促进系膜细胞增殖和分泌ECMs，可能是促进DN进展的重要机制之一。进一步，我们利用计算机软件预测了以EGR1为靶基因的microRNAs（miRs），结果发现miR-124及miR-181a等miRs可能通过作用于靶基因EGR1参与DN的肾脏纤维化进展，并在OLETF大鼠肾脏检测了其表达，证实了糖尿病大鼠肾脏miR-181a-5p表达下调，同时EGR1、TGF-β1、FN及Col I 表达上调，在高糖刺激下体外培养的HK-2细胞中miR-181a-5p与EGR1表达呈明显负相关，并且证实了miR-181a-5p通过作用于EGR1-3’-UTR直接抑制EGR1表达，进而降低TGF-β1等促纤维化因子表达。因此，miR-181a-5p通过抑制EGR1防治DN肾脏纤维化，可能是DN新的潜在的治疗靶点。