miR-155在mTOR抑制糖尿病肾病巨噬细胞自噬中的作用

Inflammatory response is an important pathogenesis of diabetic nephropathy (DN). Macrophages, as the major inflammatory cells, are closely related to the progression of DN. We found that mTOR activation reduced macrophage autophagy in DN and enhanced inflammation, which was not mediated through the classical pathway of ULK; however, the mechanism is still not clear. Furthermore, miR-155 which is regulated by mTOR is characterized by proinflammatory effect. PP2A, as a potential target gene of miR-155, may regulate autophagy through the GSK3β-WNT pathway. Therefore, it is believed that mTOR can inhibit macrophage autophagy in DN through miR-155/GSK3β-WNT pathway. In vitro study in this project detected the regulatory role of miR-155 in PP2A expression by using a dual luciferase reporter plasmid system, and investigated the effect of WNT on autophagy through the transfection of macrophages with WNT5A and WNT5A siRNA under high glucose condition. In addition, in vivo study constructed a DN model that was induced by STZ applying miR-155-/- mice and wild-type mice to observe the effect of miR-155 on autophagy. Finally, quantitative evaluation of miR-155 was conducted in clinical samples using graphene oxide to confirm the relationships of miR-155 with mTOR, mTOR, WNT and autophagy. Altogether, this study has important significance in exploring new ways of inflammation in DN and discovering novel molecular markers.

炎症反应是糖尿病肾病（DN）重要的发病机制。巨噬细胞作为主要的炎细胞，与DN进展密切相关。我们发现mTOR活化使DN巨噬细胞自噬减弱，炎症增强，但不通过ULK途径，机制尚不清楚。miR-155受mTOR调控，具有致炎作用。PP2A作为miR-155潜在靶基因可能通过GSK3β-WNT通路调节自噬。因此认为mTOR通过miR-155/GSK3β-WNT通路抑制DN巨噬细胞自噬。本课题体外研究利用双荧光酶报告质粒系统检测miR-155对PP2A的表达调控，并通过对高糖活化的巨噬细胞转染WNT5A及其 siRNA研究WNT对自噬的影响。体内研究采用miR-155-/-小鼠和野生型小鼠构建STZ诱导的DN模型，观察miR-155对自噬的调控。最后在临床样本中用氧化石墨烯定量评价miR-155，验证其与mTOR、PP2A、WNT、自噬的相关性。本研究对探索DN炎症新途径、发现新分子标志物有重要意义。

糖尿病肾病是导致终末期肾病的主要原因，高达27%的慢性肾脏疾病归因于糖尿病肾病。糖尿病患病率逐年增高，预计到2035年将超过3.5亿人，约20-40%的糖尿病患者并发糖尿病肾病。因此，寻找可应用于临床的糖尿病肾病早期诊断生物标志物，阐明其在糖尿病肾病中发病机制中的作用，意义尤为重要。本研究在体外实验，通过高糖刺激活化的人巨噬细胞THP-1，流式细胞及免疫荧光技术证实其向M1表型转分化，随着葡萄糖浓度增加，M1型细胞比例逐渐减低。利用miR-155 inhibitor和mimics对高糖刺激的THP-1人巨噬细胞进行的转染，分别下调和上调miR-155，在转录水平利用mTOR-siRNA进行转染，在蛋白水平利用雷帕霉素干预THP-1细胞，从而下调和抑制mTOR。RT-PCR证实，抑制mTOR后miR-155减少，miR-155的靶基因PP2A转录增加，导致WNT通路抑制，从而使高糖环境下的THP-1细胞自噬增强。体内实验证实，糖尿病肾病db/db小鼠肾组织中以M2为主要表型的巨噬细胞在肾血管周围及肾间质浸润。通过糖尿病肾病患者表型和病理分期的相关性研究，发现炎症急性时项蛋白触珠蛋白在糖尿病肾病患者中显著降低，并通过机器学习方法基于炎症蛋白建立糖尿病肾病诊断模型，实现有效的无创诊断。