微小RNA⁃204⁃3p通过靶向调控Krüppel样因子6对高糖诱导小鼠足细胞损伤的保护作用

Study on the protective mechanism of overexpression of microRNA⁃204⁃3p on mouse podocytes injury induced by high glucose through targeting Krüppel⁃like factor 6

目的探讨微小RNA⁃204⁃3p(miR⁃204⁃3p)对高糖诱导小鼠足细胞MPC5损伤的保护作用及其分子机制。方法采用高糖(30 mmol/L D⁃葡萄糖)处理MPC5细胞。实时定量PCR(qPCR)检测细胞中miR⁃204⁃3p与锌指蛋白Krüppel样因子6(KLF6)mRNA水平。MPC5细胞分为NG组(正常对照),HG组(高糖刺激),HG+miR⁃204⁃3p组、HG+miR⁃NC组、HG+si⁃KLF6组、HG+si⁃NC组,HG+miR⁃204⁃3p+pcDNA⁃KLF6组和HG+miR⁃204⁃3p+pcDNA组。蛋白质印迹法(Western Blot)检测肌动蛋白微丝偶联蛋白(synaptopodin)、结蛋白(desmin)、B细胞淋巴瘤/白血病⁃2(Bcl⁃2)、Bcl⁃2相关X蛋白(Bax)与活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved⁃caspase⁃3)蛋白表达,流式细胞仪检测细胞凋亡,生物学信息预测和双荧光素酶报告基因分析miR⁃204⁃3p和KLF6的靶向关系。结果与正常对照相比,高糖可显著降低MPC5细胞中miR⁃204⁃3p表达量[(0.41±0.04)比(1.02±0.08)],上调KLF6 mRNA[(2.86±0.27)比(1.03±0.08)]和蛋白[(0.89±0.08)比(0.46±0.04)]水平,还可上调KLF6、desmin[(0.81±0.07)比(0.34±0.03)]、Bax、cleaved⁃caspase⁃3蛋白表达及细胞凋亡率[(22.46±2.08)%比(5.26±0.64)%],下调synaptopodin蛋白[(0.34±0.03)比(0.76±0.07)]及Bcl⁃2蛋白表达量(P<0.05)。与HG+miR⁃NC组比较,HG+miR⁃204⁃3p组明显提高synaptopodin[(0.67±0.06)比(0.32±0.03)]及Bcl⁃2蛋白水平,降低desmin[(0.41±0.04)比(0.83±0.08)]、Bax、cleaved⁃caspase⁃3蛋白水平及细胞凋亡率[(11.25±1.65)%比(24.58±2.47)%](P<0.05)。与HG+si⁃NC组比较,HG+si⁃KLF6组明显提高synaptopodin[(0.62±0.06)比(0.28±0.03)]及Bcl⁃2蛋白水平,降低desmin[(0.49±0.05)比(0.86±0.08)]、Bax蛋白水平及细胞凋亡率[(14.69±1.87)%比(25.47±2.68)%](P<0.05)。miR⁃204⁃3p直接靶向KLF6。与HG+miR⁃204⁃3p+pcDNA组比较,HG+miR⁃204⁃3p+pcDNA⁃KLF6组显著增加高糖刺激小鼠肾脏足MPC5细胞中KLF6、desmin[(0.68±0.06)比(0.36±0.04)]、Bax蛋白表达量和细胞凋亡率[(18.41±1.67)%比(11.05±1.02)%],显著减少synaptopodin蛋白[(0.38±0.03)比(0.69±0.07)]和Bcl⁃2蛋白表达量(P<0.05)。结论miR⁃204⁃3p通过直接靶向KLF6抑制高糖刺激的足细胞凋亡,保护细胞损伤。

Objective To investigate the protective effects of microRNA⁃204⁃3p(miR⁃204⁃3p)on high glucose⁃induced mouse podocytes(MPC5)injury and its molecular mechanism.Methods MPC5 cells were treated with high glucose(30 mmol/L D⁃glu⁃cose).The levels of miR⁃204⁃3p and Krüppel⁃like factor 6(KLF6)mRNA in MPC5 cells were detected by real⁃time quantitative PCR(qPCR).MPC5 cells were assigned into NG group(normal control),HG group(high glucose stimulation),HG+miR⁃204⁃3p group,HG+miR⁃NC group,HG+si⁃KLF6 group,HG+si⁃NC group,HG+miR⁃204⁃3p+pcDNA⁃KLF6 group and HG+miR⁃204⁃3p+pcD⁃NA group.Western Blot was used to detect synaptopodin,desmin,B cell lymphoma/leukemia⁃2(Bcl⁃2),Bcl⁃2 related X protein(Bax)and activated cysteine⁃containing aspartate proteolytic enzyme⁃3(cleaved⁃caspase⁃3)protein expression.Flow cytometry was used to detect apoptosis,biological information prediction and double luciferase reporter gene analysis the targeting relationship of miR⁃204⁃3p and KLF6.Results Compared with normal controls,high glucose significantly reduced the expression of miR⁃204⁃3p in MPC5 cells[(0.41±0.04)vs.(1.02±0.08)],up⁃regulated KLF6 mRNA[(2.86±0.27)vs.(1.03±0.08)]and protein[(0.89±0.08)vs.(0.46±0.04)]levels,up⁃regulated KLF6,desmin[(0.81±0.07)vs.(0.34±0.03)],Bax,cleaved⁃caspase⁃3 protein expres⁃sion and cell apoptosis death rate[(22.46±2.08)%vs.(5.26±0.64)%],and down⁃regulated synaptopodin protein[(0.34±0.03)vs.(0.76±0.07)]and Bcl⁃2 protein expression(P<0.05).Compared with the HG+miR⁃NC group,the HG+miR⁃204⁃3p group significantly increased synaptopodin[(0.67±0.06)vs.(0.32±0.03)]and Bcl⁃2 protein levels,and decreased desmin[(0.41±0.04)vs.(0.83±0.08)],Bax,cleaved⁃caspase⁃3 protein levels and apoptosis rate[(11.25±1.65)%vs.(24.58±2.47)%](P<0.05).Compared with the HG+si⁃NC group,the HG+si⁃KLF6 group significantly increased synaptopodin[(0.62±0.06)vs.(0.28±0.03)]and Bcl⁃2 protein levels,and decreased desmin[(0.49±0.05)vs.(0.86±0.08)],Bax protein level and apoptosis rate[(14.69±1.87)%vs.(25.47±2.68)%](P<0.05).miR⁃204⁃3p directly targets KLF6.Compared with the HG+miR⁃204⁃3p+pcDNA group,the HG+miR⁃204⁃3p+pcDNA⁃KLF6 group significantly increased the ratio of KLF6 and desmin[(0.68±0.06)vs.(0.36±0.04)],Bax protein ex⁃pression and cell apoptosis rate[(18.41±1.67)%vs.(11.05±1.02)%]in the MPC5 cells,and significantly reduced synaptopodin protein[(0.38±0.03)vs.(0.69±0.07)]and Bcl⁃2 Protein expression(P<0.05).Conclusion miR⁃204⁃3p directly targets SGK1 to inhibit apoptosis of podocytes stimulated by high glucose and protects cell damage.