公方晓　孙仁华　徐云祥　呼邦传浙江省人民医院重症医学科，浙江杭州　310014

论著

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

公方晓孙仁华徐云祥呼邦传
浙江省人民医院重症医学科，浙江杭州310014

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（cyclin D1）的靶向调控作用。 方法使用LipofectamineTM2000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。Western blot检测各组细胞cyclin D1的蛋白表达情况。构建荧光素酶报告基因，内含cyclin D1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48～120 h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72 h，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48 h，miR-20a-5p组的cyclin D1蛋白表达减少（P＜0.05）。与阴性对照miRNA相比，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cyclin D1的表达，cyclin D1是miR-20a-5p的直接靶基因。

细胞周期蛋白D1；细胞增殖；肾小管；微RNAs

［Abstract］Objective To observe the effect ofmiR-20a-5p's on the proliferation of human renal tubular epithelial cells and to verify its targeted regulation of cyclin D1.M ethods miRNAswere transfected into HK-2 cells（human renal tubular epithelial cell）by LipofectamineTM2000.Cellswere divided into three groups，themiR-20a-5p group transfected with miR-20a-5p，the negative control group transfected with negative control miRNA and the blank control group transfected with nomiRNAs.The absorbances of cells in each group after transfection were evaluated by Cell Counting 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 ofmiR-20a-5p and 3'UTR.Results The absorbances ofmiR-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 G1stage inmiR-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 inmiR-20a-5p group was lower than that of the other two groups at 48 h after transfection（P＜0.05）.Compared with negative controlmiRNA，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.Themechanism may bemiR-20a-5p can repress expression of cyclin D1 and cyclin D1 is the direct target gene ofmiR-20a-5p.

［Key words］Cyclin D1；Cell proliferation；Kidney tubules；microRNAs

急性肾损伤（acute kidney injury，AKI）是重症患者中的一种常见合并症，在ICU中的发病率约20%～60%，住院死亡率常超过25%，死亡率较高［1，2］。根据改善全球肾脏病预后组织（KDIGO）2012年临床实践指南［3］，AKI的定义为：48 h内血清肌酐上升≥26.5μmol/L；或7 d内血清肌酐上升至≥1.5倍基线水平；或连续6 h尿量＜0.5mL/（kg·h）。AKI的病理生理过程中，肾小管上皮细胞损伤是重要的病理性变化。……