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.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2024 Dec 25;53(6):756-764. doi: 10.3724/zdxbyxb-2024-0504.
To study the role of microRNA (miR)-30d-5p in high glucose-induced podocyte injury.
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.
Under high glucose induction, podocyte apoptosis increased, miR-30d-5p and P62 expressions were upregulated, while nephrin, ATG5, PINK1, PARK2 and LC3Ⅱ/LC3Ⅰ expressions decreased (all <0.01). MiR-30d-5p inhibitor reversed the effect of high glucose on apoptosis, and the expression of ATG5, PINK1, PARK2, nephrin, LC3Ⅱ/LC3Ⅰ and P62 (all <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 <0.05).
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.
研究微小RNA（miR）-30d-5p在高糖诱导的足细胞损伤中的作用。
用30 mmol/L葡萄糖使足细胞发生糖基化，转染miR-30d-5p抑制剂和模拟物，然后用1 mg/mL 3-甲基腺嘌呤（3-MA）处理。通过逆转录PCR定量miR-30d-5p的转染效率。用流式细胞术检测细胞凋亡。通过蛋白质免疫印迹法检测nephrin、微管相关蛋白轻链（LC）3Ⅱ/LC3Ⅰ、P62、自噬相关基因（ATG）5、PTEN诱导的推定激酶（PINK）1和Parkin基因（PARK2）的表达。用JC-1荧光探针检测线粒体膜电位，并用相关试剂盒检测细胞中的三磷酸腺苷（ATP）含量。
在高糖诱导下，足细胞凋亡增加，miR-30d-5p和P62表达上调，而nephrin、ATG5、PINK1、PARK2和LC3Ⅱ/LC3Ⅰ表达下降（均<0.01）。miR-30d-5p抑制剂逆转了高糖对细胞凋亡的影响，以及ATG5、PINK1、PARK2、nephrin、LC3Ⅱ/LC3Ⅰ和P62的表达（均<0.01）。高糖诱导足细胞线粒体膜电位丧失和ATP含量降低，而抑制miR-30d-5p则使其增加。自噬抑制剂3-MA和miR-30d-5p模拟物逆转了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<0.05）。
抑制miR-30d-5p可能通过促进ATG5、PINK1、PARK2的表达并减轻高糖诱导的足细胞损伤来促进线粒体自噬（线粒体吞噬）。
