1,25(OH)_(2)D_(3)对miR-130b转染的高糖条件下HMC细胞TGF-β1/Smad3及Col-Ⅳ表达的影响

Effect of 1,25(OH)_(2)D_(3) on the Expression of TGF-β1/Smad3 and Col-Ⅳ in HMC Cells Transfected with miR-130b Under High Glucose

目的观察高糖环境下1,25(OH)_(2)D_(3)对miR-130b转染肾小球系膜细胞TGF-β1/Smad3、Col-IV表达水平的变化,探索1,25(OH)_(2)D_(3)是否可通过miR-130b改善糖尿病肾病肾脏纤维化的过程。方法HMC细胞传代培养,miR-130b mimics及miR-130b inhibitor经LipofectamineTM 2000转染肾小球系膜细胞,依据分组加入1,25(OH)_(2)D_(3)1.0×10^(-8) mol/L,37℃、5%CO_(2)饱和湿度条件下继续培养48 h,共分为6组:①空白对照组;②细胞+无水乙醇组(A组);③细胞+miR-130b mimics+无水乙醇组(B组);④细胞+miR-130b mimics+1,25(OH)_(2)D_(3)组(C组);⑤细胞+miR-130b inhibitor+无水乙醇组(D组);⑥细胞+miR-130b inhibitor+1,25(OH)_(2)D_(3)组(E组)。以实时荧光定量PCR检测细胞miR-130b及TGF-β1、Smad3、Col-IVmRNA的表达水平,以Western blot方法检测细胞TGF-β1、Smad3、Col-IV蛋白的表达水平。采用多组间比较的单因素方差分析及组内两两比较采用LSD、Dunnett s T3法比较各组数据。结果空白对照组与A组细胞miR-130b、TGF-β1、Smad3、Col-IV表达水平,差异无统计学意义(P>0.05),与A组相比,B组miR-130b、TGF-β1、Smad3、Col-IV表达水平明显升高(P<0.05),D组miR-130b、TGF-β1、Smad3、Col-IV表达水平明显降低(P<0.05)。经1,25(OH)_(2)D_(3)治疗发现,与B组相比,C组miR-130b、TGF-β1、Smad3、Col-IV表达水平明显降低(P<0.05),与D组相比,E组miR-130b、TGF-β1、Smad3、Col-IV表达水平明显降低(P<0.05)。结论1,25(OH)_(2)D_(3)可降低miR-130b转染肾小球系膜细胞miR-130b及纤维化相关因子TGF-β1、Smad3、Col-IV的表达水平,1,25(OH)_(2)D_(3)可能通过miR-130b改善糖尿病肾病肾脏纤维化的过程。

Objective To investigate the effect of 1,25(OH)_(2)D_(3) on the expression of TGF-β1/Smad3 and Col-IV in glomerular mesangial cells transfected with miR-130b in high glucose environment,and to explore whether 1,25(OH)_(2)D_(3) can improve the process of renal fibrosis in diabetic nephropathy through miR-130b.Methods HMC cells were subcultured,and the miR-130b mimics and miR-130b inhibitor were transfected into glomerular mesangial cells by lipofectamineTM 2000.According to the experimental design,HMC cells were stimulated with 1,25(OH)_(2)D_(3)1.0×10^(-8) mol/L at 37℃and 5%CO_(2) saturation humidity for 48h and were divided into six groups:①blank control group;②cells+absolute ethanol group(group A);③cells+miR-130b mimics+absolute ethanol group(group B);④cells+miR-130b mimics+1,25(OH)_(2)D_(3) group(group C);⑤cell+miR-130b inhibitor+absolute ethanol group(group D);⑥cell+mir-130b inhibitor+1,25(OH)_(2)D_(3) group(group E).The mRNA expression levels of miR-130b,TGF-β1,Smad3 and Col-IV were detected by real-time PCR,and the protein expression levels of TGF-β1,Smad3 and Col-IV were detected by Western blot.LSD and Dunnett s T3 were used to analyze the data.Results It showed that there was no significant difference in the expression levels of miR-130b,TGF-β1,Smad3 and Col-IV between the blank control group and group A(P>0.05).Compared with group A,the expression levels of miR-130b,TGF-β1,Smad3 and Col-IV in group B were significantly increased(P<0.05).The expression levels of miR-130b,TGF-β1,Smad3 and Col-IV were significantly decreased in group D(P<0.05).After 1,25(OH)_(2)D_(3) treatment,the expression levels of miR-130b,TGF-β1,Smad3 and Col-IV in group C were significantly lower than those in group D(P<0.05),and the expression levels of miR-130b,TGF-β1,Smad3 and Col-IV in group E were significantly lower than those in group D(P<0.05).Conclusion 1,25(OH)_(2)D_(3) can reduce the expression levels of miR-130b and fibrosis related factors TGF-β1,Smad3 and Col-IV in glomerular mesangial cells transfected with miR-130b,and may improve the process of renal fibrosis in diabetic nephropathy through miR-130b.