Klotho通过抑制高糖诱导的微RNA-21a-5p高表达而减轻肾小管上皮细胞间质转分化的机制

Mechanism of Klotho reducing the mesenchymal transdifferentiation of renal tubular epithelial cells by inhibiting the increased expression of microRNA-21a-5p induced by high glucose

目的探索Klotho抑制高糖条件下肾小管上皮细胞(HK-2)微RNA(miR)-21a-5p表达而减轻肾小管上皮细胞间质转分化的机制。方法将体外培养的HK-2分为低糖组、高糖组(30 mmol/L葡萄糖)和高渗组(30 mmol/L甘露醇);按照高糖刺激时间分为4组:0、12、24、48 h;按照是否转染pcDNA3.1-Klotho/pcDNA3.1-Vector及转染后低或高糖刺激分为4组:低糖组、高糖组、高糖+pcDNA3.1-Vector组和高糖+pcDNA3.1-Klotho组;按照转染si-Klotho/si-Negative后是否加入二硫代氨基甲酸吡咯烷(PDTC)和低或高糖刺激分为6组:低糖+si-Klotho组、低糖+si-Negative组、低糖+si-Klotho+PDTC组、高糖+si-Klotho组、高糖+si-Negative组和高糖+si-Klotho+PDTC组。染色质免疫共沉淀(CHIP)分组:低糖+pcDNA3.1-Vector组、高糖+pcDNA3.1-Vector组和高糖+pcDNA3.1-Klotho组。采用实时聚合酶链式反应检测各组细胞miR-21a-5p水平;Western blot检测Klotho、纤连蛋白(FN)、α-平滑肌动蛋白(α-SMA)、核因子κB(NF-κB)、NF-κB 抑制因子(IκB)表达水平;CHIP检测NF-κB对miR-21a-5p启动子的直接作用。两组间比较采用t检验,多组间比较采用单因素方差分析。结果与低糖组比较,高糖组Klotho蛋白表达下降至60.8%(P<0.05),miR-21a-5p表达升高至3.203倍(P<0.01),FN和α-SMA表达明显升高。随着高糖刺激时间延长,Klotho蛋白表达水平逐渐降低,而miR-21a-5p表达水平逐渐升高,呈现相反的变化趋势(P<0.05)。高糖组核蛋白NF-κB和miR-21a-5p表达水平是低糖组的2.934倍和3.154倍(均P<0.01),而过表达Klotho后,NF-κB和miR-21a-5p表达水平分别下降至高糖组的50.18%和49.24%(均P<0.05)。CHIP结果显示:高糖+pcDNA3.1-Vector组与低糖+pcDNA3.1-Vector组相比,NF-κB和miR-21a-5p启动子结合显著增加至3.121倍(1.000±0.742比3.121±0.115,P<0.01);而过表达Klotho能显著减弱高糖引起的NF-κB和miR-21a-5p启动子结合至54.21%(3.121±0.115比1.692±0.073,P<0.01)。结论 Klotho可抑制高糖条件下肾小管上皮细胞NF-κB和miR-21a-5p启动子的结合,使miR-21a-5p表达减少,从而减轻肾小管上皮细胞间质转分化。

Objective To explore the mechanism of Klotho reducing the mesenchymal transdifferentiation of renal tubular epithelial cells(HK-2)by inhibiting the expression of microRNA(miR)-21a-5p in HK-2 under high glucose conditions.Methods HK-2 cells cultured in vitro were divided into low glucose(LG)group,high glucose(HG)group(30 mmol/L glucose),and hyperosmolar(HM)group(30 mmol/L mannitol).According to the time of high glucose stimulation,HK-2 cells were divided into 0,12,24 and 48 h.According to pcDNA3.1-Klotho/pcDNA3.1-Vector transfection and low/high glucose stimulation,HK-2 cells were divided into:LG group,HG group,HG+pcDNA3.1-Vector group and HG+pcDNA3.1-Klotho group.According to si-Klotho/si-Negative transfection and low/high glucose stimulation and whether adding pyrrolidine dithiocarbamate(PDTC),HK-2 cells were divided into sixgroups:LG+si-Klotho group,LG+si-Negative group,LG+si-Klotho+PDTC group,HG+si-Klotho group,HG+si-Negative group and HG+si-Klotho+PDTC group.Chromatin immunoprecipitation(CHIP)test groups included:LG+pcDNA3.1-Vector group,HG+pcDNA3.1-Vector group and HG+pcDNA3.1-Klotho group.The expression of miR-21a-5p was detected by real-time polymerase chain reaction;the protein expression of Klotho,fibronectin(FN),α-smooth actin(α-SMA),nuclear factor kappa-B(NF-κB)and NF-κB inhibitor(IκB)were evaluated by Western blot;the direct effect of NF-κB on the promoter of miR-21a-5p was detected by CHIP.The t test analysis was used for comparison between two groups,and one-way ANOVA was used for comparison among multiple groups.Results Compared with the LG group,the protein expression of Klotho in the HG group was decreased to 60.8%(P<0.05),the expression of miR-21a-5p was increased to 3.203 times(P<0.01),and the expression of FN andα-SMA were increased significantly.With the prolonged time of high glucose stimulation,the expression of Klotho was gradually decreased,while the expression of miR-21a-5p was gradually increased,the trend was in the opposite direction(P<0.05).Compared with the LG group,the expression levels of nucleoprotein NF-κB and miR-21a-5p in the HG group were increased to 2.934 times and 3.154 times(both P<0.01)respectively;while overexpression of Klotho can reduce the expression of NF-κB and miR-21a-5p to 50.18%and 49.24%(both P<0.05)of the HG group.The CHIP results showed that compared with the LG+pcDNA3.1-Vector group,the combination of NF-κB and miR-21a-5p promoter was increased to 3.121 times(1.000±0.742 vs 3.121±0.115,P<0.01)in the HG+pcDNA3.1-vector group;while overexpression of Klotho can significantly reduce the high combination of NF-κB and miR-21a-5p promoter to 54.21%(3.121±0.115 vs 1.692±0.073,P<0.01).Conclusion Klotho can reduce the mesenchymal transdifferentiation of renal tubular epithelial cells by inhibiting the binding of NF-κB and miR-21a-5p promoter and reducing the expression of miR-21a-5p under high glucose conditions.