线粒体未折叠蛋白反应通过LncRNA Tug1改善糖尿病肾病肾小管损伤的作用及机制研究

Diabetic nephropathy is a serious complication of diabetes mellitus which is lack of effective treatment. And renal tubular injury is considered as an important pathological basis for its progression. Now mitochondria dysfunction is regard as key point of tubular injury. Our previous study found maintaining UPRmt activity in high glucose stimulation could improve the expression of LncRNA Tug1 and Mfn2，reduce tubular injury. However, the mechanism about it is unknown. Bioinformatics indicated that miR-520f-3p may be the connection between LncRNA Tug1 and Mfn2. In view of this, by means of molecular biology experience, this project will take renal tubular cells as research object and create DN rat model to detect whether UPRmt improve renal tubular injury by up-regulating Mfn2 expression. Furthermore, we will investigate the role of LncRNA Tug1 in UPRmt regulating Mfn2 expression. And based on the above experiments, we further analyze the possible way in which LncRNA Tug1 participates in the regulation of UPRmt to improve renal tubular mitochondrial damage. The smoothly development of this project will contribute to clarify the pathogenesis and provide scientific basis for the prevention and treatment of diabetic nephropathy.

糖尿病肾病是糖尿病严重的并发症，目前缺乏有效的治疗手段，而肾小管损伤是其发生及进展的重要病理基础。现已知线粒体受损是介导肾小管损伤的关键。申请人前期研究发现高糖环境中维持线粒体未折叠蛋白反应（UPRmt）活性可促进LncRNA Tug1及线粒体融合蛋白Mfn2表达，减轻肾小管细胞损伤，但其机制不明。生物信息学提示miR-520f-3p可能是连接LncRNA Tug1及Mfn2的桥梁。为此本项目拟以肾小管上皮细胞为研究对象，同时建立糖尿病肾病大鼠模型，通过分子生物学实验，首先阐明UPRmt反应是否通过上调Mfn2表达改善肾小管损伤，其次明确LncRNA Tug1在UPRmt反应上调Mfn2表达改善小管损伤中的作用，最后揭示LncRNA Tug1参与调控UPRmt反应改善DN肾小管损伤的可能机制。该项目的完成有助于进一步阐明DN的发病机制，为DN的防治提供新的理论及实验依据。

糖尿病肾病是糖尿病严重的并发症，目前缺乏有效的治疗手段。肾小管损伤是其发生及进展的重要病理基础。我们在DN肾小管线粒体损伤中，线粒体未折叠蛋白反应活性降低。利用Hsp60过表达质粒构建完成高UPRmt反应活性的HK-2细胞，成功构建Mfn2 siRNA和过表达质粒，通过干扰线粒体融合蛋白 Mfn2 的表达，证实UPRmt反应可上调线粒体融合蛋白Mfn2表达发挥改善DN肾小管损伤的作用。高糖诱导肾小管损伤过程中，UPRmt反应活性改变可影响LncRNA Tug1的表达及细胞内亚定位分布，我们进一步构建LncRNA Tug1慢病毒过表达质粒及siRNA，发现LncRNA Tug1表达上调可改善高糖诱导的HK-2细胞线粒体形态和功能及细胞损伤情况，并上调线粒体融合蛋白Mfn2的表达。我们通过生物信息学查找结果提示，miR-520f-3p是LncRNA Tug1及Mfn2的共同miRNA。为进一步阐明LncRNA Tug1 调控 UPRmt 反应改善 DN 肾小管损伤的作用机制，我们进一步构建miR-520f-3p mimics或inhibitor，证实干扰 miR-520f-3p 表达，可上调Mfn2水平，同时改善高糖诱导的肾小管线粒体及细胞损伤，并证实LncRNA Tug1可通过miR-520f-3p发挥其生物学作用。进一步通过构建STZ诱导的DN大鼠，尾静脉注射Hsp60 慢病毒过表达质粒或LncRNA Tug1慢病毒过表达载体，证实UPRmt反应对DN肾小管损伤的保护作用。研究结论：UPRmt反应通过LncRNA Tug1/miR-520f-3p/Mfn2有效改善DN肾小管线粒体和细胞损伤。