抑制微RNA-30d-5p促进线粒体自噬缓解高糖作用下的足细胞损伤  

Inhibition of miR-30d-5p promotes mitochondrial autophagy and alleviates high glucose-induced injury in podocyte

作　　者：蔡莹 陈生[1] 蒋晓立 邬启元 郭蓓[1] 王芳[1] CAI Ying;CHEN Sheng;JIANG Xiaoli;WU Qiyuan;GUO Bei;WANG Fang(Department of Nephrology,Ningbo Medical Center Lihuili Hospital,Ningbo 315000,Zhejiang Province,China)

机构地区：[1]宁波市医疗中心李惠利医院肾内科,浙江宁波315000

出　　处：《浙江大学学报(医学版)》2024年第6期756-764,共9页Journal of Zhejiang University(Medical Sciences)

基　　金：宁波市自然科学基金(2021J282)。

摘　　要：目的:探讨微RNA(miR)-30d-5p在高糖诱导下足细胞损伤中的作用。方法:采用30 mmol/L葡萄糖诱导足细胞高糖化,使用miR-30d-5p抑制剂和模拟物转染,再经1 mg/mL 3-甲基腺嘌呤(3-MA)处理。定量逆转录聚合酶链反应检测miR-30d-5p的转染效率;流式细胞术检测细胞凋亡;蛋白质印迹法检测肾病蛋白、微管相关蛋白轻链(LC)3Ⅱ/LC3Ⅰ、P62、自噬相关基因(ATG)5蛋白、PTEN诱导假定激酶(PINK)1和Parkin基因(PARK2)蛋白表达。JC-1作为荧光探针检测线粒体膜电位,通过相关试剂盒检测细胞中的三磷酸腺苷(ATP)含量。结果:在高糖诱导下,足细胞凋亡增加,miR-30d-5p和P62表达上调,肾病蛋白、ATG5、PINK1、PARK2和LC3Ⅱ/LC3Ⅰ表达水平降低(均P<0.01)。miR-30d-5p抑制剂部分逆转了高糖对足细胞凋亡以及ATG5、PINK1、PARK2、肾病蛋白、LC3Ⅱ/LC3Ⅰ和P62表达的影响(均P<0.01)。高糖诱导足细胞线粒体膜电位流失、ATP含量减少,抑制miR-30d-5p后能增加膜电位和ATP含量(均P<0.05)。自噬抑制剂3-MA和miR-30d-5p模拟物可逆转miR-30d-5p抑制对高糖诱导的足细胞凋亡、自噬和线粒体功能的影响(均P<0.05)。结论:抑制miR-30d-5p可能通过促进ATG5、PINK1、PARK2的表达促进线粒体自噬,进而缓解高糖诱导的足细胞损伤。Objective:To study the role of microRNA(miR)-30d-5p in high glucoseinduced podocyte injury.Methods:Podocytes were hyperglycated with 30 mmol/L glucose,transfected with miR-30d-5p inhibitor and mimic,and then treated with 1 mg/mL 3-methyladenine(3-MA).The transfection efficiency of miR-30d-5p was quantified by reverse transcription PCR.Apoptosis was detected by flow cytometry.The expressions of nephrin,microtubule-associated protein light chain(LC)3Ⅱ/LC3Ⅰ,P62,autophagy-related gene(ATG)5,PTEN induced putative kinase(PINK)1 and Parkin gene(PARK2)were detected by Western blotting.The mito-chondrial membrane potential was detected by JC-1 fluorescent probe,and adenosine triphosphate(ATP)content in cells was detected by relevant kits.Results:Under high glucose induction,podocyte apoptosis increased,miR-30d-5p and P62 expressions were upregulated,while nephrin,ATG5,PINK1,PARK2 and LC3Ⅱ/LC3Ⅰexpressions decreased(all P<0.01).MiR-30d-5p inhibitor reversed the effect of high glucose on apoptosis,and the expression of ATG5,PINK1,PARK2,nephrin,LC3Ⅱ/LC3Ⅰand P62(all P<0.01).High glucose induced loss of mitochondrial membrane potential and ATP content in podocytes,while inhibition of miR-30d-5p increased them.Autophagy inhibitors 3-MA and miR-30d-5p mimics reversed the effects of miR-30d-5p inhibition on apoptosis,autophagy and mitochondrial function of podocytes induced by high glucose(all P<0.05).Conclusions:Inhibition of miR-30d-5p may promote mitochondrial autophagy(mitophagy)by promoting the expression of ATG5,PINK1,PARK2 and alleviating high glucose-induced podocyte damage.

关 键 词：糖尿病肾病 miR-30d-5p 高糖 足细胞 细胞凋亡 自噬 线粒体功能 

分 类 号：R587.2[医药卫生—内分泌]