南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (12): 1839-1845.doi: 10.12122/j.issn.1673-4254.2022.12.12

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miR-34a通过靶向抑制Notch信号通路减轻糖尿病肾病小鼠的足细胞损伤

王欢岚，刘 红，张燕敏，陈伟栋   

  1. 武汉市第一医院（武汉市中西医结合医院）肾内科，湖北 武汉 430022
  • 出版日期:2022-12-20 发布日期:2023-01-12

MiR-34a alleviates podocyte injury in mice with diabetic nephropathy by targeted downregulation of Notch signaling pathway

WANG Huanlan, LIU Hong, ZHANG Yanmin, CHEN Weidong   

  1. Department of Nephrology, Wuhan First Hospital (Wuhan Hospital of Integrated Traditional Chinese and Western Medicine), Wuhan 430022, China
  • Online:2022-12-20 Published:2023-01-12

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摘要： 目的 探讨miR-34a介导Notch信号通路对糖尿病肾病（DN）足细胞损伤及凋亡的影响。方法 体外实验：通过RT-PCR法检测高糖（30 mmol/L）环境下足细胞中miR-34a表达水平，构建miR-34a过表达足细胞系（miR-34a组）及其阴性对照（miR-NC组），使用荧光素酶实验验证miR-34a与Notch 1的靶向关系，构建Notch 1过表达足细胞系（Notch 1组）和miR-34a、Notch 1均表达升高细胞系（miR-34+Notch 1组）；采用CCK-8法检测足细胞存活情况，流式细胞法检测细胞凋亡情况，Western blot法检测细胞凋亡关蛋白水平。体内实验：通过高脂饮食和链脲佐菌素建立DN小鼠模型并分为模型组、miR-34a组（n=15/组），另选15只不做干预小鼠为对照组，miR-34a组和模型组小鼠分别尾静脉注射agomir-34a[80 mg/(kg·d)]、agomir-NC[80 mg/(kg·d)]，连续注射3 d，4周后，HE染色、TUNEL法分别观察肾组织病理、凋亡情况，Western blot检测肾组织凋亡相关蛋白和Notch 1蛋白表达水平。结果 高糖环境下足细胞中miR-34a表达水平低于正常糖诱导下足细胞和高渗透压下作用下的足细胞（P<0.05），miR-34a组足细胞中Notch 1表达水平低于miR-NC组（P<0.05）；Notch 1 wt/miR-34a组荧光素酶活力值低于Notch 1 wt组（P<0.05），而Notch 1 mut/miR-34a组和Notch 1 mut/miR-NC组荧光素酶活力值差异无统计学意义（P>0.05）；miR-34a组足细胞的A值高于miR-NC组（P<0.05），而细胞凋亡率和caspase-3、caspase-9、Bax/Bcl-2蛋白水平均低于miR-NC组（P<0.05）；Notch 1组足细胞的A值低于miR-NC组（P<0.05），而细胞凋亡率和caspase-3、caspase-9、Bax/Bcl-2蛋白水平均高于miR-NC组（P<0.05）；miR-34a+Notch 1组足细胞的A值低于miR-34a组（P<0.05），而细胞凋亡率和caspase-3、caspase-9、Bax/Bcl-2蛋白水平均高于miR-34a组（P<0.05）。对照组小鼠肾组织结构清晰完整，模型组小鼠肾小球肿胀、体积增大，miR-34a组小鼠肾组织病变程度得到改善；模型组小鼠肾组织 miR-34a 表达水平低于对照组（P<0.05），miR-34a 组小鼠的肾组织 miR-34a 表达水平高于对照组（P<0.05）；模型组小鼠肾组织凋亡指数和caspase-3、caspase-9、Notch 1蛋白水平高于对照组（P<0.05），miR-34a组小鼠肾组织凋亡指数和caspase-3、caspase-9、Notch 1蛋白水平低于模型组（P<0.05）。结论 miR-34a可通过靶向作用Notch 1改善DN足细胞损伤和凋亡。

关键词: 糖尿病肾病；miR-34a；Notch信号通路；足细胞；凋亡

Abstract: Objective To explore the effects of miR-34a on injury and apoptosis of podocytes in diabetic nephropathy (DN) and the role of Notch signaling pathway in mediating its effects. Methods The expression of miR-34a in podocytes exposed to high glucose (30 mmol/L) was detected using RT-PCR. A podocyte line with miR-34a overexpression was constructed, and the miRNA-target relationship between miR-34a and Notch 1 was verified with luciferase assay. The effects of overexpression of Notch 1 and both miR-34a and Notch 1 on podocyte survival and apoptosis were evaluated using CCK-8 and flow cytometry and by detecting apoptosis- related proteins using Western blotting. In a DN mouse model established by high- fat diet and streptozotocin, the effect of tail vein injection of agomir-34a and agomir-NC on pathology and apoptosis in the renal tissues were observed with HE staining and TUNEL staining, and the renal expressions of apoptosis-related proteins and Notch 1 protein were detected with Western blotting. Results High glucose exposure significantly lowered miR-34a expression in cultured human podocytes (P<0.05). The expression of Notch 1 was significantly lowered in miR-34a-overexpressing podocytes as compared with the cells with miR-NC transfection (P<0.05). Luciferase assay confirmed the mRNA-target relationship between miR-34a and Notch 1 (P<0.05). MiR-34a overexpression obviously promoted podocyte survival (P<0.05), reduced Notch 1 expression, and lowered apoptosis rate and the protein expressions of caspase-3, caspase- 9 and Bax/Bcl-2 levels in the cells (P<0.05), while the reverse changes were observed in Notch 1-overexpressing podocytes (P<0.05). In DN mouse models, treatment with miR-34a obviously alleviated renal pathologies. Compared with that in the control group, the expression level of miR-34a in the renal tissues was significantly lowered in DN model group (P<0.05) and increased in miR-34a group (P<0.05). The mice in the model group showed significantly higher apoptosis index of the renal tissues with increased expressions of caspase-3, caspase-9 and Notch 1 (P<0.05), which were lowered by treatment with miR-34a (P<0.05). Conclusion MiR-34a is capable of improving podocyte injury and apoptosis in DN mice by targeted downregulation of Notch 1.

Key words: diabetic nephropathy; miR-34a; notch signaling pathway; podocyte; apoptosis