miR-20a-5p通过调控细胞周期蛋白D1抑制肾小管上皮细胞增殖

miR-20a-5p inhibits proliferation of renal tubular epithelial cells by targeting cyclin D1

目的观察miR-20a-5p对肾小管上皮细胞增殖的影响，并验证其对细胞周期蛋白D1（cyclinD1）的靶向调控作用。方法使用Lipofectamine^TM 2000脂质体将微小RNA（miRNA）转染入人肾小管上皮细胞HK-2，实验分为3组。miR-20a-5p组转染miR-20a-5p，对照组转染阴性对照miRNA，空白组未做任何转染。CCK-8实验检测转染后各组细胞的吸光度值，流式细胞术检测各组的细胞周期变化。Westernblot检测各组细胞cyclinD1的蛋白表达情况。构建荧光素酶报告基因，内含cyclinD1的3’端非翻译区（3’UTR），双荧光素酶报告基因实验验证miR-20a-5p对3’UTR的直接结合作用。结果MiR-20a-5p组在转染后48—120h的吸光度值均低于对照组和空白组（P〈0．05），另外两组差异无统计学意义（P〉0.05）。转染后72h，miR-20a-5P组处于G1期的比例为（63．89±3．61）％，高于另外两组（P〈0．05），而另外两组差异无统计学意义（P〉0．05）。转染后48h，miR-20a-5p组的cyclinD1蛋白表达减少（P〈0．05）。与阴性对照miRNA相此，miR-20a-5p能抑制荧光素酶的活性（P〈0．05）。结论miR-20a-5p能抑制肾小管上皮细胞的增殖，并将细胞阻滞在G1期，其可能机制是miR-20a-5p抑制cyclinD1的表达，cyclinD1是miR-20a-5p的直接靶基因。

Objective To observe the effect of miR-20a-5p＇s on the proliferation of human renal tubular epithelial cells and to verify its targeted regulation of cyclin D1. Methods miRNAs were transfected into HK-2 cells（human renal tubular epithelial cell）＇by LipofectamineTM2000. Cells were divided into three groups, the miR-20a-5p group transfected with miR-20a-5p, the negative control group transfected with negative control miRNA and the blank control group transfected with no miRNAs. The absorbances of cells in each group after transfection were evaluated by Cell C6unting Kit-8 （CCK-8）, the cell cycles of each group were assayed by flow cytometry. The protein expression of cyclin D1 in each group after transfection was detected via Western blot. A firefly luciferase reporter vector containing 3＇ UTR of cyclin D1 was created and the Dual-Luciferase Reporter Assay System was used to test the direct combination of miR- 20a-5p and 3＇UTR. Results The absorbances of miR-20a-5p group were lower than those of negative control group and blank control group at 48-120 h after transfection （P〈0.05）, and there was no statistical difference between the other two groups（P〉0.05）. The percentage of G1 stage in miR-20a-5p group was（63.89±3,61）% at 72 h after transfection, higher than those of the other two groups（P〈0.05）, and there was no statistical difference between the other two groups （P〉0.05）. The protein expression of cyclin D1 in miR-20a-5p group was lower than that of the other two groups at 48 h after transfection（P〈0.05）. Compared with negative control miRNA, miR-20a-5p repressed the luciferase activity（P〈0.05）. Conclusion miR-20a-5p can inhibit the proliferation of human renal tubular epithelial cells and cause a delay in the G1-stage. The mechanism may be miR-20a-5p can repress expression of cyclin D1 and cyclin D1 is the direct target gene of miR-20a-5p.