miR-346在糖尿病肾病TGF-β介导的Smad信号通路中的作用

Role of miR-346 in TGF-β-mediated Smad Signaling Pathway in the Pathogenesis of Diabetic Nephropathy

目的:观察miR-346在高糖环境下小鼠肾小球系膜细胞TGF-β介导Smad信号通路中的作用,探讨miR-346参与糖尿病肾病(DN)的发病机制。方法:体外高糖(25 mmol/L)下培养小鼠肾小球系膜细胞(MC),p Genesil-miR-346和重组TGF-β药物作为干预因素,分别设TGF-β药物刺激组(10、30 ng/m L)、TGF-β药物刺激+shRNA P53干扰组、阴性对照组。细胞免疫荧光检测细胞表型;Western法检测P53蛋白和Smad3/4蛋白的表达;实时荧光定量RT-PCR法检测mRNA表达。结果:⑴miR-346的表达可由P53通过调节miR-346基因中启动子来调节;⑵miR-346可调节Smad3/4的表达,其过程是TGF-β通过P53、miR-346通路从上游来调节的。结论:miR-346通过TGF-β/Smad信号通路调控糖尿病肾病相关基因表达,二者形成正反馈效应,在DN的发生和发展中起到非常重要的作用。

Objective （1）To investigate the role of miR-346 in TGF-β-mediated Smad signaling pathway in mouse mesangial cells under high glucose concentrations;（2）to explore the molecular mechanism of miR-346 in the occurrence and devel- opment of diabetic nephropathy.Methods Mouse mesangial cells（MCs） were cultured in vitro and treated with high glucose concentration at dosage of 25 mmol/L.Then pGenesil-miR-346 and recombinant TGF-β drugs were applied as intervention factors.MCs were divided into recombinant TGF-β drug stimulation group （ 10,30 ng/mL） ,recombinant TGF-β drug stim- ulation＋P53 RNA interference group,negative control group.Immunofluorescence was appliedto detect the cell phenotype; Western blot was used to detect the protein expression level of P53 and Smad3/4 ; Real-time RT-PCR was performed to de- tect the expression level of mRNA.Results （1） P53 could regulate the promoter of miR-346 to control the expression of miR -346.（2）miR-346 could regulate the protein expression of Smad3/4, which was performed by TGF-β through the upstream of P53-miR-346 signaling.Conclusion miR-346 could regulate the diabetic nephropathy-related gene expression through TGF-β/Smad signaling pathway via a positive feedback effect,which may play an important role in the occurrence and de- velopment of diabetic nephropathy.