miRNA-30a-5p过表达对肾癌细胞株786-0增殖、凋亡的影响

Effects of overexpression of miRNA-30a-5p on proliferation and apoptosis of renal carcinoma cell line 786-0

目的观察miRNA-30a-5p过表达对肾癌细胞株786-0增殖、凋亡的影响,并探讨其机制。方法将肾癌细胞株786-0分为模拟物组、抑制物组、未转染组,模拟物组转染miRNA-30a-5p模拟物,抑制物组转染miRNA-30a-5p抑制物,未转染组为正常肾癌细胞株786-0。采用实时荧光定量PCR法检测模拟物组、抑制物组、未转染组肾癌细胞株786-0及正常人肾小管上皮细胞HK-2(正常组)miRNA-30a-5p,CCK-8法观察各组培养24、48、72 h后的细胞增殖能力,流式细胞术观察前三组细胞凋亡情况,Western blotting法检测前三组细胞中异黏蛋白(MTDH)表达。结果正常组、模拟物组、抑制物组、未转染组细胞miRNA-30a-5p mRNA相对表达量分别为1.00±0.00、1.32±0.05、0.32±0.05、0.52±0.06,正常组、模拟物组、抑制物组与未转染组相比,P均<0.01。正常组在培养24、48、72 h后的吸光度值分别为0.26±0.02、0.44±0.03、0.73±0.06,模拟物组分别为0.33±0.04、0.50±0.05、0.88±0.05,抑制物组分别为0.86±0.01、1.88±0.12、2.48±0.15,未转染组分别为0.62±0.01、1.63±0.02、2.34±0.23,正常组、模拟物组各时点分别与抑制物组、未转染组相比,P均<0.01。模拟物组、抑制物组、未转染组细胞凋亡率分别为11.91%±0.45%、5.23%±0.38%、6.77%±0.52%,模拟物组与抑制物组、未转染组相比,P均<0.01。模拟物组、抑制物组、未转染组MTDH蛋白的相对表达量分别为1.14±0.08、6.31±0.24、5.56±0.06,模拟物组与抑制物组、未转染组相比,P均<0.01。结论 miRNA-30a-5p过表达可明显抑制肾癌细胞株786-0的增殖、促进细胞凋亡,其可能是通过调控MTDH蛋白表达实现的。

Objective To observe the influence of overexpression of miRNA-30a-5p on the proliferation and apoptosis of renal carcinoma cell line 786-0,and to explore the mechanism.Methods The renal carcinoma 786-0 cells were divided into the mimic group,the inhibitor group,and the non-transfected group.The cells in the mimic group were transfected with miRNA-30a-5p mimics,cells in the inhibitor group were transfected with miRNA-30a-5p inhibitor,and in the nontransfected group,the cells were normal renal carcinoma 786-0 cells.The expression of miRNA-30a-5p mRNA in the mimics group,inhibitor group,non-transfected group,and normal human renal tubular epithelial cells HK-2（ normal group）were detected by real-time fluorescence quantitative PCR; the cell proliferation at 24,48,and 72 h after transfection was evaluated by using CCK-8 assay; the apoptosis of the former three groups was assessed by flow cytometry; the expression of MTDH protein in the former three groups was detected by Western blotting.Results The miRNA-30a-5p mRNA expression in the normal group,mimic group,inhibitor group,and non-transfected group was 1.00 ± 0.00,1.32 ± 0.05,0.32± 0.05,and 0.52 ± 0.06,respectively; significant difference was found between the normal group,mimic group,inhibitor group,and the non-transfected group（ all P〈0.01）.The absorbance values of the normal group at 24,48,and 72 h were0.26 ± 0.02,0.44 ± 0.03,and 0.73 ± 0.06,respectively; the absorbance values of the mimic group were 0.33 ± 0.04,0.50 ± 0.05,and 0.88 ± 0.05,respectively; in the inhibitor group they were 0.86 ± 0.01,1.88 ± 0.12,and 2.48 ±0.15,respectively; in the non-transfected group,they were 0.62 ± 0.01,1.63 ± 0.02,and 2.34 ± 0.23,respectively;significant difference was found between the normal group,the mimic group,and the inhibitor group and the non-transfected group at each time point,all P〈0.01.The apoptosis rates in the mimic group,inhibitor group,and non-transfected group were separately 11.91% ± 0.45%,5.23% ± 0.38%,and 6.77% ± 0.52%; significant difference was found between the mimic group and between the inhibitor group and non-transfected group,all P〈0.01.The expression levels of MTDH protein in the mimic group,inhibitor group,and non-transfected group were separately 1.14 ± 0.08,6.31 ± 0.24,and 5.56 ± 0.06,and significant difference was found in the expression of MTDH protein of the mimic group as compared with that of the inhibitor group and non-transfected group,all P〈0.01.Conclusion Overexpression of miRNA-30a-5p may inhibit 786-0 cell proliferation and promote the apoptosis by regulating the expression of MTDH.