下调微小RNA-21增强肾癌对舒尼替尼敏感性的作用及其机制

Inhibition of microRNA-21 sensitizes renal cell carcinoma to sunitinib by targeting phosphatase and tensin homologue deleted on chromosome ten

目的:探讨下调微小RNA(miRNA,miR)-21与肾癌细胞舒尼替尼敏感性的相关性及其机制。方法:应用实时定量反转录聚合酶链反应(RT-qPCR)检测人肾癌细胞株786-O、ACHN和正常肾小管上皮细胞株HK-2中miR-21的表达。脂质体Lipofectamine 2000瞬时转染miR-21抑制剂及阴性对照无义序列至肾癌细胞,噻唑蓝(MTT)法检测细胞活性,并计算转染前后舒尼替尼24 h半数抑制浓度(IC 50)。RT-qPCR及蛋白质印迹法(Western blot)验证miR-21对第10号染色体上缺失与张力蛋白同源的磷酸酯酶基因(PTEN)表达的影响。应用SPSS 20.0和GraphPad prism 7.0软件分析,两组间比较采用 t检验。 结果:与HK-2比较,miR-21在786-O(0.005±0.001比1.001±0.063, t=27.269, P<0.01)、ACHN(0.005±0.001比0.257±0.060, t=7.218, P<0.01)中呈显著高表达。舒尼替尼对转染前后786-O、ACHN的24 h IC 50分别为16.52、4.79 μmol/L;15.31、3.66 μmol/L。miR-21抑制剂组PTEN mRNA表达水平显著高于空白对照组(786-O:5.295±1.088比1.005±0.119, t=6.789, P<0.01;ACHN:3.248±0.720比1.017±0.220, t=5.135, P<0.01)及无义对照序列组(786-O:5.295±1.088比0.966±0.212, t=6.763, P<0.01;ACHN:3.248±0.720比0.933±0.175, t=5.414, P<0.01),PTEN蛋白表达差异无统计学意义,但均出现一定程度升高。 结论:下调miR-21通过调控抑癌基因PTEN表达,影响肾癌细胞对舒尼替尼敏感性,并且能使舒尼替尼在一个较低浓度下发挥抑制肾癌细胞活性作用。

Objective To investigate the effect and mechanism of microRNA(miRNA,miR)-21 down-regulation on sunitinib sensitivity of renal cell carcinoma(RCC).Methods The miR-21 expression level of RCC cell lines 786-O,ACHN and renal tubular epithelial cell line HK-2 was detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).The anti-miR-21 and scrambled sequence were transfected into 786-O and ACHN cells with Lipofectamine 2000.After transfection,methyl thiazol tetrazolium(MTT)assay was used to evaluate the cell viability and the half maximal inhibitory concentration(IC50)was calculated afterwards.Last,RT-qPCR and Western blotting were performed to validate the inhibition of anti-miR-21 transfection on phosphatase and tensin homologue deleted on chromosome ten(PTEN)expression.All analyses were carried out by SPSS 20.0 and GraphPad prism 7.0 software.The statistical significance was assessed with t-test.Results The expression level of miR-21 in 786-O(1.001±0.063 vs.0.005±0.001,t=27.269,P<0.01)or ACHN cells(0.257±0.060 vs.0.005±0.001,t=7.218,P<0.01)was significantly higher than in HK-2 cells.The 24 h IC50 of sunitinib in 786-O(16.52μmol/L)or ACHN(15.31μmol/L)cells before anti-miR-21 transfection was obviously higher than that in respective cells after transfection(4.79 and 3.66μmol/L).As compared with blank control group(for 786-O:1.005±0.119 vs.5.295±1.088,t=6.789,P<0.01;for ACHN:1.017±0.220 vs.3.248±0.720,t=5.135,P<0.01)or scrambled sequence-transfected group(for 786-O:0.966±0.212 vs.5.295±1.088,t=6.763,P<0.01;for ACHN:0.933±0.175 vs.3.248±0.720,t=5.414,P<0.01),RT-qPCR revealed a significant increase in PTEN mRNA levels in anti-miR-21-transfected group.A considerable,even though not significant,increase in PTEN protein expression was observed in anti-miR-21-transfected group.Conclusion These findings demonstrated that the sunitinib sensitivity of RCC could be modulated through miR-21-mediated regulation of PTEN.