糖尿病肾病相关微小RNA对高糖下肾小球系膜细胞增殖的影响及其机制

目的探讨在高糖培养条件下,糖尿病肾病相关微小RNA(MicroRNA,miRNA)即miR-21对小鼠肾小球系膜细胞生长、增殖的影响,及miR-21高表达对PTEN/PI3K信号途径的影响。方法采用高糖培养的小鼠肾小球系膜细胞模拟糖尿病状态,在Lipofectamine2000介导下经miR-21真核表达质粒pGenesil-miR-21转染,G418筛选获得稳定表达细胞株。采用实时RT-PCR检测转染细胞miR-21的表达水平,MTT法检测细胞增殖,细胞免疫化学法和Western blot法检测PTEN/PI3K信号途径关键分子PTEN、phospho-Ak(tSer473)和PI3Kp85α蛋白的表达水平。结果筛选出的肾小球系膜细胞能稳定高表达miR-21;高表达的miR-21对细胞增殖能力有明显的抑制作用,且可显著下调PTEN的表达水平(P<0.05),同时上调phospho-Akt(Ser473)和PI3Kp85α的表达水平(P<0.05)。结论 miR-21可通过特异性靶向抑制PTEN蛋白的表达,提高phospho-Akt(Ser473)和PI3Kp85α蛋白的表达水平,以减缓肾小球系膜细胞增殖,可能为一个预防和治疗糖尿病肾病新型的潜在靶点。

Effect of Diabetic Nephropathy-associated microRNA on Mesangial Cell Proliferation Induced by High Glucose and Relevant Mechanism

Objective To investigate the effect of diabetic nephropathy(DN)-associated microRNA miR-21 on the growth and proliferation of mesangial cells at high glucose concentration as well as the effect of high expression of miR-21 on PTEN / PI3K signal pathway. Methods Mouse mesangial cells were cultured at high glucose concentration to mimic diabetes mellitus, and transfected with eukaryotic expression vector pGenesil-miR-21 in mediation of Lipofectamine 2000. The cell clones for stable expression were screened with G418. The expression level of miR-21 in transfected cells was determined by real-time RT-PCR, and the cell prolifera- tion by MTT method. The expressions of PTEN, phospho-Akt (Ser473)and PI3K p85α proteins, the key molecules of PTEN / PI3K signal pathway, were determined by immunocytochemical method. Results The miR-21 was expressed highly and stably in the screened mesangial cells. The highly expressed miR-21 significantly inhibited the proliferation of mesangial cells and down-regulated the expression of PTEN(P < 0. 05), while significantly up-regulated the expressions of phospho-Akt(Ser473)and PI3K p85α(P < 0. 05). Conclusion The miR-21 may increase the expression levels of phospho-Akt(Ser473)and PI3K p85α proteins by specific inhibiting the expression of PTEN protein, thus inhibit the proliferation of mesangial cells, which may be a novel potential target for prevention and therapy of DN.