miR-155表达变化对TGF-β1损伤足细胞蛋白podocin、CD2AP、synaptopodin的影响

Effects of miRNA-155 expression changes on TGF-β1 injured podocyte proteins podocin,CD2AP and synaptopodin

目的:研究miR-155 mimics/inihibitor转染足细胞后微小RNA-155(miR-155)的表达变化及其对TGF-β1损伤足细胞的影响。方法:体外培养小鼠足细胞,细胞免疫荧光染色鉴定足细胞分化成熟;用TGF-β1处理足细胞构建足细胞损伤模型,RT-PCR和Western blot分别检测miR-155和CD2AP的表达;免疫荧光显微镜观察转染成功指示剂Cy3,RT-PCR检测miR-155 mimics/miR-155 inihibitor转染(0 h、24 h、48 h、72 h)后miR-155的表达;选取转染时间48~72 h,RT-PCR检测Normal、TGF-β1、TGF-β1+mimics、TGF-β1+mimics NC、TGF-β1+inhibitor、TGF-β1+inhibitor NC组细胞miR-155及podocin、CD2AP、synaptopodin的mRNA表达,Western blot检测3种蛋白的表达,免疫荧光观察各组CD2AP的荧光分布和表达。结果:CD2AP呈足细胞分化成熟的特征分布;TGF-β1干预后miR-155表达上升,CD2AP蛋白表达下降(P<0.05);miR-155 mimics或miR-155 inihibitor转染48 h、72 h后,miR-155表达明显增加或减少(P<0.05);miR-155 mimics可上调TGF-β1诱导的miR-155表达(P<0.05),且可促进TGF-β1诱导的足细胞podocin、CD2AP、synaptopodin的mRNA和蛋白表达下降(P<0.05),而转染miR-155 inihibitor对足细胞蛋白的作用与miR-155 mimics相反。免疫荧光结果显示CD2AP蛋白的表达与Western blot、RT-PCR结果一致。结论:miR-155可靶向足细胞蛋白podocin、CD2AP和synaptopodin的表达,miR-155过表达可促进足细胞损伤,miR-155表达下调则可改善足细胞的损伤,因此,miR-155可作为足细胞损伤新的药物靶标。

Objective:To investigate the expression changes of microRNA-155(miR-155)and its effect on TGF-β1-injured podocytes after miR-155 mimics/inihibitor transfected podocytes.Methods:Cultured mouse podocytes in vitro and identified the podocyte differentiation and maturation by immunofluorescence staining;podocytes were treated with TGF-β1 to construct a podocyte injury model,and the expressions of miR-155 and CD2 AP were detected by RT-PCR and Western blot,respectively;immunofluorescence microscopy was used to detect the success indicator Cy3.RT-PCR was used to detect expression of miR-155 after miR-155 mimics/miR-155 inihibitor transfection of mouse glomerular podocyte cell(0 h,24 h,48 h,72 h).Select transfection time 48~72 h,to detected mRNA expressions of miR-155 and podocin,CD2 AP,synaptopodin in normal,TGF-β1,TGF-β1+mimics,TGF-β1+mimics NC,TGF-β1+inhibitor,and TGF-β1+inhibitor NC group by RT-PCR.The expression of three proteins was detected by Western blot,and the fluorescence distribution and expression of CD2 AP in each group were observed by immunofluorescence.Results:CD2 AP showed a characteristic distribution of podocyte differentiation and maturation.The miR-155 expression was increased and CD2 AP protein expression was decreased after intervention with TGF-β1(P<0.05).After transfection with miR-155 mimics or miR-155 inihibitor for48 h and 72 h,The expression of miR-155 was increased or decreased significantly(P<0.05);miR-155 mimics can up-regulate the miR-155 expression induced by TGF-β1(P<0.05),and can promote the reduction of podocyte podocin,CD2 AP,synaptopodin mRNA and protein expression induced by TGF-β1(P<0.05),while The effect of miR-155 inihibitor on podocyte proteins was opposite to that of miR-155 mimics.The immunofluorescence results showed that the expression of CD2 AP protein was consistent with the results of Western blot and RT-PCR.Conclusion:miR-155 can target the expression of podocyte proteins podocin,CD2 AP and synaptopodin.Overexpression of miR-155 can promote podocyte injury.Down-regulation of miR-155 can improve podocyte injury.miR-155 can be used as a new drug target for podocyte injury.