miR-451对肾小球系膜细胞增殖的抑制作用及其机制

Inhibitory effect of miR-451 on proliferation of mesangial cells

目的探讨微小RNA(microRNA,miRNA)-451对肾小球系膜细胞增殖的抑制作用及其机制。方法在Lipofectamine 2000介导下,将miR-451模拟物(miR-451 mimics)及mimics对照分别转染高糖培养的肾小球系膜细胞,另设高糖未转染组和低糖未转染组,实时荧光RT-PCR法检测各组细胞中miR-451的表达水平;MTT法检测miR-451对小鼠肾小球系膜细胞增殖的影响;Western blot法检测靶蛋白Ywhaz、p-p38 MAPK和p-MKK3的表达水平。结果 miR-451组细胞中miR-451的表达水平较mimics对照组明显升高(P<0.01);miR-451组第2、3、4天的细胞增殖能力明显低于高糖未转染组(P均<0.05),第3、4天明显低于mimics对照组(P均<0.05);与mimics对照组和高糖未转染组比较,miR-451组细胞中Ywhaz、p-p38 MAPK和p-MKK3的表达水平均明显下降(P均<0.05)。结论 miR-451可抑制Ywhaz蛋白的表达,降低p38 MAPK信号途径中p-p38 MAPK和p-MKK3蛋白的表达,从而降低高糖诱导的肾小球系膜的增殖。本实验为研究糖尿病肾病(diabetic nephropathy,DN)的发病机制提供了新的思路。

Objective To investigate the inhibitory effect miR-451, a diabetic nephropathy （DN）-associated microRNA （miRNA） on mesangial cell proliferation induced by high glucose concentration as well as the relevant mechanism. Methods Mouse mesangial cells were cultured at high glucose concentration to mimic diabetes mellitus and transfected with miR-451 mimics and mimics control in mediation of Lipofectamine 2000, using those untransfected and those transfected after culture at low glucose concentration as control. The expression of miR-451 gene in cells of various groups were determined by RT-PCR, while the cell proliferation by MTT assay, and for expression levels of Ywhaz, p-p38 MAPK and p-MKK3 by Western blot. Results The expression level of miR-451 gene in ceils of miR-451 group was significantly higher than that in mimics control group （P 〈 0. 01 ）. As compared with those in miR-451 group, the proliferation levels of untransfeeted cells cultured at high glucose concentration on days 2, 3 and 4 as well as those in mimics control group on days 3 and 4 increased significantly （each P 〈 0. 05 ）. However, compared with those in mimics control group and those in the untransfected cells cultured at high glucose concentration, the expression levels of Ywhaz, p-p38 MAPK and p-MKK3 decreased significantly （each P 〈 0. 05）. Conclusion The miR-451 inhibited the expression of Ywhaz protein specifi- cally, while decreased the expression levels of p-p38 MAPK and p-MKK3 in p38 MAPK signal pathway, thus inhibited the proliferation of mesangial cells induced by high glucose concentration. It provided an experimental basis for the study on molecular mechanism of DN.