【摘要】 目的探讨微小RNA-135b(miR-135b)在糖尿病肾病(DN)发病中的作用及可能机制。方法应用荧光定量PCR及原位杂交检测miR-135b在DN肾组织中的表达。采用生物信息学预测miR-135b的靶基因,并通过蛋白免疫印迹法及检测双荧光素酶报告基因活性进行验证。同时行蛋白免疫印迹法检测DN系膜细胞增殖及系膜基质相关标志蛋白的表达。结果实时定量PCR显示,miR-135b在DN肾组织中表达较正常肾组织升高(P<0.05)。原位杂交显示,miR-135b在DN肾组织中表达较正常肾组织增强,且其表达主要位于肾小球系膜细胞及肾小管上皮细胞的胞浆和胞核中。人肾小管上皮细胞HMC及人系膜细胞HK-2在高糖培养24 h时均能上调miR-135b的表达,48 h升高更显著,与同时点正常对照培养的HMC及HK-2比较差异均有统计学意义(P均<0.05)。人源和鼠源的Smad5 3’-非翻译区与miR-135b种子序列的8个碱基UUUCGGUA完全互补。miR-135b模拟物可下调Smad5的蛋白表达水平,而miR-135b抑制物对Smad5的蛋白表达无影响。将Smad5 3’-非翻译区野生型和突变型质粒分别转染入HMC及HK-2,并同时转染miR-135b模拟物,发现miR-135b模拟物能抑制Smad5 3’-非翻译区野生型荧光素酶活性,但对Smad5 3’-非翻译区突变体的荧光素酶活性无影响。miR-135b促进系膜细胞增殖及系膜基质增加HMC细胞转染miR-135b模拟物,可有效下调其靶蛋白Smad5的表达,同时上调增殖核抗原(PCNA)和细胞周期蛋白(Cyclin D1)表达,且使细胞周期蛋白依赖性激酶的抑制蛋白p21表达降低(P均<0.05),但转染miR-135b抑制物对PCNA、Cyclin D1、p21的表达无影响(P均>0.05)。结论 MiR-135b通过调控Smad5基因促进系膜细胞增殖及肾脏纤维化。更多还原

【Abstract】 Objective To investigate the effect and potential mechanism of microRNA-135b(miR-135b) on the pathogenesis of diabetic nephropathy(DN).Methods The expression of miR-135 b in the kidney tissue of DN patients was quantitatively measured by fluorescent quantitative PCR and in situ hybridization.The target gene of miR-135 b was predicted by bioinformatics method,which was validated by Western blot and double luciferase reporter activity.Western blot was adopted to detect the expression of marker proteins related to mesangial cell proliferation and mesangial matrix.Results Real-time quantitative PCR revealed that the expression of miR-135 b in the kidney tissue of DN patients was significantly up-regulated compared with that in the normal kidney tissue(P < 0.05).In situ hybridization demonstrated that the expression of miR-135 b in the DN kidney tissue was up-regulated than that in the normal kidney tissue.It was mainly distributed in the glomerulus mesangial cells and the cytoplasm and nucleus of renal tubular epithelial cells.The expression levels of miR-135 b were up-regulated in the HMC and HK-2 cultured in high-glucose medium for 24 h,and more significantly enhanced for 48 h,which significantly differed from those in HMC and HK-2 in the control group(all P< 0.05).Human and mouse Smad5 3’-untranslated region completely complemented with the 8 bases of UUUCGGUA in the miR-135 b seed sequence.MiR-135 b simulant could down-regulate the expression level of Smad5 protein,whereas miR-135 b inhibitor exerted no effect upon the expression of Smad5 protein.HMC and HK-2 cells were transfected with wild-type and mutant Smad5 3’-untranslated region vectors,and simultaneously transfected with miR-135 b simulant.MiR-135 b simulant could inhibit the activity of fluorescein enzyme of wild-type Smad5 3’-untranslated region,whereas exerted no effect upon the activity of mutant Smad5 3’-untranslated region.MiR-135 b could promote mesangial cell proliferation and increased mesangial matrix.HMC cells transfected with miR-135 b simulant could effectively down-regulate the expression of target protein Smad5,upregulate the expression of proliferating cell nuclear antigen(PCNA) and Cyclin D1 and down-regulate the expression of cyclin-dependent kinase inhibitor p21 protein(all P < 0.05).However,transfection with miR-135 b inhibitor exerted no effect upon the expression of PCNA,Cyclin D1 and p21(all P > 0.05).Conclusion MiR-135 b promotes mesangial cell proliferation and kidney fibrosis through regulating Smad5 gene.更多还原