[微小RNA-34a通过靶向下调Notch信号通路减轻糖尿病肾病小鼠的足细胞损伤]
[MiR-34a alleviates podocyte injury in mice with diabetic nephropathy by targeted downregulation of Notch signaling pathway].
作者信息
Wang H, Liu H, Zhang Y, Chen W
机构信息
Department of Nephrology, Wuhan First Hospital (Wuhan Hospital of Integrated Traditional Chinese and Western Medicine), Wuhan 430022, China.
出版信息
Nan Fang Yi Ke Da Xue Xue Bao. 2022 Dec 20;42(12):1839-1845. doi: 10.12122/j.issn.1673-4254.2022.12.12.
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 ( < 0.05). The expression of Notch 1 was significantly lowered in miR-34a-overexpressing podocytes as compared with the cells with miR-NC transfection ( < 0.05). Luciferase assay confirmed the mRNA-target relationship between miR-34a and Notch 1 ( < 0.05). MiR-34a overexpression obviously promoted podocyte survival ( < 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 ( < 0.05), while the reverse changes were observed in Notch 1-overexpressing podocytes ( < 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 ( < 0.05) and increased in miR-34a group ( < 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 ( < 0.05), which were lowered by treatment with miR-34a ( < 0.05).
CONCLUSION
MiR-34a is capable of improving podocyte injury and apoptosis in DN mice by targeted downregulation of Notch 1.
目的
探讨微小RNA-34a（miR-34a）对糖尿病肾病（DN）足细胞损伤和凋亡的影响以及Notch信号通路在介导其作用中的作用。
方法
采用逆转录聚合酶链反应（RT-PCR）检测高糖（30 mmol/L）处理的足细胞中miR-34a的表达。构建miR-34a过表达的足细胞系，通过荧光素酶报告基因实验验证miR-34a与Notch 1之间的miRNA-靶标关系。采用细胞计数试剂盒-8（CCK-8）法、流式细胞术以及蛋白质免疫印迹法检测凋亡相关蛋白，评估Notch 1过表达以及miR-34a和Notch 1同时过表达对足细胞存活和凋亡的影响。在高脂饮食联合链脲佐菌素建立的DN小鼠模型中，通过苏木精-伊红（HE）染色和TUNEL染色观察尾静脉注射miR-34a激动剂和阴性对照激动剂对肾组织病理学和凋亡的影响，并用蛋白质免疫印迹法检测肾组织中凋亡相关蛋白和Notch 1蛋白的表达。
结果
高糖处理显著降低了培养的人足细胞中miR-34a的表达（P<0.05）。与转染miR-NC的细胞相比，miR-34a过表达的足细胞中Notch 1的表达显著降低（P<0.05）。荧光素酶报告基因实验证实了miR-34a与Notch 1之间的mRNA-靶标关系（P<0.05）。miR-34a过表达明显促进足细胞存活（P<0.05），降低Notch 1表达，降低细胞凋亡率以及半胱天冬酶-3（caspase-3）、半胱天冬酶-9（caspase-9）和Bax/Bcl-2蛋白的表达水平（P<0.05），而Notch 1过表达的足细胞则出现相反的变化（P<0.05）。在DN小鼠模型中，miR-34a处理明显减轻了肾脏病理改变。与对照组相比，DN模型组肾组织中miR-34a的表达水平显著降低（P<0.05），miR-34a组则升高（P<0.05）。模型组小鼠肾组织凋亡指数显著升高，caspase-3、caspase-9和Notch 1的表达增加（P<0.05），miR-34a处理后这些指标降低（P<0.05）。
结论
miR-34a可通过靶向下调Notch 1改善DN小鼠足细胞损伤和凋亡。
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