人肾脏系膜细胞内mRNA稳定因子HuR蛋白对细胞周期蛋白cyclinD1转录后表达的调节

The mRNA-stabilizing factor Human-Antigen R regulates renal cyclinD1 post-transcriptional expression in human mesangial cells

目的观察人肾脏系膜细胞在血管紧张素Ⅱ(AngⅡ)刺激下mRNA稳定因子人类抗原R(HuR)蛋白对细胞周期蛋白D1(cyclinD1)蛋白的作用。方法体外培养人肾脏系膜细胞,根据AngⅡ对其的刺激时间即0、3、6、9、24 h分别记为0A、3A、6A、9A、24A组。应用流式细胞仪检测0A组与24A组细胞周期;免疫细胞化学方法观察各组HuR蛋白的亚细胞定位变化;Western blot方法检测各组全细胞与胞质内HuR蛋白及全细胞cyclinD1表达水平;RT-PCR方法检测各组cyclinD1的RNA水平。干扰RNA方法观察抑制HuR蛋白表达后AngⅡ刺激下对全细胞cyclinD1表达的保护效应。结果与0A组比较,24A组细胞增殖趋势明显;HuR蛋白亚细胞定位从胞核转移到胞质,其中3A组变化最显著;全细胞HuR蛋白表达水平不变,胞质内HuR蛋白表达增强,其中3A组最显著(P<0.05);cyclinD1蛋白表达增强,其中24A组最显著(P<0.05),而RNA水平保持不变(P>0.05)。与转染前相比,干扰RNA转染后3A组胞质HuR蛋白表达明显减弱(P<0.05);24A组cyclinD1蛋白表达明显减弱(P<0.05),而RNA水平不变(P>0.05)。结论①AngⅡ可刺激人肾小球系膜细胞增殖;②AngⅡ刺激下人肾脏系膜细胞核内HuR蛋白向胞质内转移;③HuR蛋白参与AngⅡ刺激下cyclinD1蛋白表达过程。

Objective To investigate the role of mRNA stabilizing factor Human-Antigen R （HuR） in angiotensin 1I （AnglI）-stimulated cyclinD1 over-expression in cultured human mesangial cells. Methods Human mesangial cells cultured in vitro were divided into the 0A group,3A group, 6A group, 9A group and 24A group according to the time gradient of angiotensin II management（0 h,3 h,6 h,9 h,and 24 h）. The cell cycle was detected by flow cytometry and subcellular localization of HuR protein was determined by imrnunochemistry. Total cyclinD1 protein , cytoplasmic and total HuR protein expression were detected by Western blot and total cyclinD1 mRNA level was determined by RT- PCR. Using RNA interference technology to down regulate HuR protein level, we surveyed the protective effect of an- giotensin 11 on total cyclinD1 protein expression. Results Compared with the 0A group, there was a marked increase in mesangial cells proliferation in the 24A group （ P 〈 0.05 ）. The HuR protein shifted from the nucleus to the cyto- plasm, especially in the 3A group. There was no significant variation of total HuR protein expression but an increase in cytoplasmic HuR protein especially in 3A group（P 〈 0.05 ）. An increase in total cyclinD1 protein expression was ob- served in the 24A group（ P 〈 0.05 ）, but there was no change in the RNA level. Compared with transfection before, there was a marked decrease in cytoplasmic HuR protein expression in the 3A group（ P 〈 0.05 ） and total cyclinD1 pro- tein expression in the 24A group （ P 〈 0.05 ）, but no change in the cyclinD1 RNA level. Conclusion （1）Ang I1 inducesproliferation of human mesangial cells. （2）HuR protein shifts from the nucleus to the cytoplasm induced by Ang II. （3） HuR protein is involved in expression of cyclinD1 protein with Ang II stimulation.