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.
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。
Keywords: apoptosis; diabetic nephropathy; miR-34a; notch signaling pathway; podocyte.