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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Lipofectamine TM2000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Mi R-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。
Objective To observe the effect of miR-20a-5p on the proliferation of renal tubular epithelial cells and to verify its targeting effect on cyclin D1. Methods MicroRNA (miRNA) was transfected into human renal tubular epithelial cells HK-2 using Lipofectamine TM 2000 liposome. The experiment was divided into three groups. The miR-20a-5p group transfected with miR-20a-5p and the control group transfected with negative Control miRNA, blank group did not make any transfection. The CCK-8 assay was used to detect the absorbance of each group after transfection, and the cell cycle changes of each group were detected by flow cytometry. Western blot was used to detect the protein expression of cyclin D1 in each group. The luciferase reporter gene was constructed and contained 3 ’untranslated region (3’UTR) of cyclin D1. Dual luciferase reporter assay was used to verify the direct binding of miR-20a-5p to 3’UTR. Results The absorbance values of Mi R-20a-5p group at 48-120 h after transfection were lower than those in control group and blank group (P <0.05). There was no significant difference between the other two groups (P> 0.05). At 72 h after transfection, the percentage of miR-20a-5p group in G1 phase was (63.89 ± 3.61)% higher than the other two groups (P <0.05), while the other two groups showed no significant difference (P> 0.05) . At 48 h after transfection, the expression of cyclin D1 in miR-20a-5p group decreased (P <0.05). Compared with the negative control miRNA, miR-20a-5p inhibited luciferase activity (P <0.05). Conclusions miR-20a-5p can inhibit the proliferation of renal tubular epithelial cells and arrest the cells in G1 phase. The possible mechanism is that miR-20a-5p inhibits the expression of cyclin D1, and cyclin D1 is a direct target of miR-20a-5p gene.