miR-93-5p靶向DUSP8基因对糖尿病肾病足细胞损伤的影响

The Effects of MiR-93-5p on Podocyte Damage in Diabetic Nephropathy by Targeting DUSP8 Gene

目的:观察miR-93-5p、双特异性磷酸酶8(Dual specificity phosphatase 8,DUSP8)对高糖诱导的人肾小球足细胞(HPC)损伤的影响,并探究其机制。方法:25mM高糖处理HPC细胞6、12、24 h,检测细胞的炎性因子白介素6(interleukin-6,IL-6)、肿瘤坏死因子α(Tumor Necrosis Factor-alpha,TNF-α)的分泌,细胞凋亡率及凋亡相关蛋白分裂的半胱氨酸天冬氨酸蛋白酶3(Cleaved caspase-3)、Bcl-2相关X蛋白(Bcl-2 Associated X Protein,Bax)、B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)的表达,筛选最适处理时间12h。将miR-con组(转染miR-con)、miR-93-5p组(转染miR-93-5p mimics)、anti-miR-con组(转染anti-miR-con)、anti-miR-93-5p组(转染anti-miR-93-5p mimics)、高糖(HG)+si-con组(转染si-con)、HG+si-DUSP8组(转染si-DUSP8)、HG+miR-93-5p+pcDNA组(共转染miR-93-5p mimics和pcDNA)、HG+miR-93-5p+pcDNA-DUSP8组(共转染miR-93-5p mimics和pcDNA-DUSP8)用脂质体法转染至HPC细胞或高糖处理12 h的HPC细胞。分别使荧光定量聚合酶链式反应(Fluorescence quantitative polymerase chain reaction,qRT-PCR)实验、免疫印迹(Western blotting,WB)实验、酶联免疫吸附(Enzyme linked immunosorbent assay,ELISA)实验、流式细胞术检测细胞miR-93-5p、DUSP8的表达、IL-6、TNF-α的分泌、DUSP8、Cleaved caspase-3、Bax、Bcl-2的蛋白表达、细胞凋亡率;双荧光素酶报告实验检测细胞的荧光活性。结果:与低糖(NG)组相比,HG(6、12、24h)组细胞miR-93-5p的表达显著降低,DUSP8表达显著升高,IL-6、TNF-α的含量显著升高,Cleaved caspase-3、Bcl-2的蛋白表达均显著降低,Bax的蛋白表达显著升高,细胞凋亡率显著升高,最适处理时间为12 h。过表达miR-93-5p抑制高糖诱导的HPC细胞IL-6、TNF-α分泌和凋亡率,并促进Cleaved caspase-3、Bcl-2蛋白,抑制Bax蛋白。miR-93-5p抑制野生型DUSP8细胞的荧光活性。沉默DUSP8具有与过表达miR-93-5p类似的保护足细胞损伤作用,过表达DUSP8则逆转过表达miR-93-5p对损伤足细胞IL-6、TNF-α分泌和凋亡的抑制作用。结论:miR-93-5p抑制高糖诱导足细胞炎性因子的分泌和凋亡,其机制与miR-93-5p靶向DUSP8相关。

Objective:To observe the effects of miR-93-5p and dual-specificity phosphatase 8(DUSP8)on high glucose-induced HPC cells,and explore its mechanism.Methods:HPC cells were treated with 25mM high glucose for 6,12,and 24 hours.The optimal treatment time was 12h by the secretion of the inflammatory factors interleukin-6(IL-6)and Tumor Necrosis Factor-alpha(TNF-α)in the cells,apoptosis rate and apoptosis-related protein split Cleaved caspase-3(Cleaved caspase-3),Bcl-2 associated X protein(Bax),B-cell lymphoma-2(Bcl-2).The miR-con group(transfected miR-con),miR-93-5p group(transfected miR-93-5p mimics),anti-miR-con group(transfected anti-miR-con),anti-miR-93-5p group(transfected anti-miR-93-5p mimics),high glucose(HG)+si-con group(transfected si-con),HG+si-DUSP8 group(transfected si-DUSP8),HG+miR-93-5p+pcDNA group(co-transfected miR-93-5p mimics and pcDNA),HG+miR-93-5p+pcDNA-DUSP8 group(co-transfected miR-93-5p mimics and pcDNA-DUSP8)were all transfected to HPC cells with liposome methods.Fluorescence quantitative polymerase chain reaction(qRT-PCR),Western blotting(WB),Enzyme linked immunosorbent assay(ELISA),flow cytometry were used to detect the expression of miR-93-5p,DUSP8,cell secretion of IL-6,TNF-α,and protein expression of DUSP8,Cleaved caspase-3,Bax,Bcl-2,and cell apoptosis rate;Dual luciferase reporter experiment detects the fluorescence activity of cells.Results:Compared with the low glucose(NG)group,the expression of miR-93-5p was significantly decreased,the expression of DUSP8 was substantially increased,the levels of IL-6 and TNF-αwere substantially increased,protein expression of Cleaved caspase-3,Bcl-2 were significantly decreased,Bax protein expression was significantly increased,the cell apoptosis rate was markedly increased in the HG(6,12,24 h)group.The optimal treatment time was 12 h.Overexpression miR-93-5p depressed the secretion of IL-6,TNF-α,apoptosis rate,and facilitated proteins of Cleaved caspase-3 and Bcl-2,and suppressed Bax protein in HPC cells induced by high glucose.MiR-93-5p inhibited the fluorescent activity of wild-type DUSP8 cells.Silencing DUSP8 had a protective effect similar to that of overexpression miR-93-5p,and overexpression DUSP8 reversed the effects of overexpression miR-93-5p on the inhibition of IL-6 and TNF-αsecretion and apoptosis in injured podocytes.Conclusion:MiR-93-5p could inhibit the secretion and apoptosis of inflammatory factors in podocytes induced by high glucose,and its mechanism is related to miR-93-5p targeting DUSP8.