摘要
目的本研究主要探讨微小RNA-122a(microRNA-122a,miR-122a)在人喉癌细胞对多西他赛(Docetaxel,DOC)敏感性中的作用和作用机制。方法慢病毒转染法上调人喉癌Hep-2细胞中miR-122a水平并设为过表达组,转染相应空载体作为空载对照组,以未转染细胞作为空白对照组;实时荧光定量聚合酶链反应(RTFQ-PCR)检测细胞miR-122a水平。DOC处理细胞,四甲基偶氮唑蓝(MTT)法检测细胞活力并计算DOC半数抑制浓度(IC50);结晶紫染色检测细胞贴壁存活能力。蛋白质印迹法(Western blot)和免疫荧光实验检测细胞p-EGFR和p-AKT蛋白表达。结果过表达组细胞miR-122a表达水平明显高于空载对照组(P<0.001)。与空载对照组比较,DOC对过表达组细胞的IC50值明显降低(P<0.001)。DOC(5.56μg/mL)处理24 h后,过表达组细胞贴壁存活率明显低于空载对照组(P=0.001)。过表达组细胞p-EGFR和p-AKT相对蛋白表达水平明显低于空载对照组(均P<0.001)。与空载对照组比较,过表达组细胞p-EGFR和p-AKT荧光强度明显降低(均P<0.001)。结论miR-122a可能通过抑制EGFR-AKT信号通路转导,从而增强人喉癌细胞对DOC的敏感性。
Objective To explore the role and mechanism of microRNA-122a(miR-122a)in the sensitivity of human laryngeal cancer cells to Docetaxel(DOC).Methods The lentiviral transfection method was used to up-regulate the level of miR-122a in human laryngeal carcinoma Hep-2 cells and it was set as an overexpression group.The corresponding empty vector was transfected as an empty control group,and the untransfected cells was set as a blank control group.Real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)was used to detect the level of miR-122a in cells.Cells were treated with DOC,and the cell viability was detected by methyl thiazolyl tetrazolium(MTT)method and the median inhibitory concentration(IC50)of DOC was calculated.The crystal violet staining was used to detect the viability of cell adhesion.The western blot and immunofluorescence experiments were used to detect the expression of p-EGFR and p-AKT proteins.Results The expression level of miR-122a in the overexpression group was significantly higher than that in the no-load control group(P<0.001).Compared with the no-load control group,the IC50 value of DOC to overexpression group cells was significantly reduced(P<0.001).After 24 hour of DOC(5.56μg/mL)treatment,the adherent survival rate of the overexpression group was significantly lower than that of the no-load control group(P=0.001).The relative protein expression levels of p-EGFR and p-AKT in the overexpression group were significantly lower than those in the no-load control group(P<0.001).Compared with the no-load control group,the fluorescence intensity of p-EGFR and p-AKT in the overexpression group was significantly reduced(P<0.001).Conclusion miR-122a may inhibit EGFR-AKT signaling pathway transduction,thereby enhancing the sensitivity of human laryngeal cancer cells to DOC.
出处
《浙江临床医学》
2023年第4期491-494,共4页
Zhejiang Clinical Medical Journal
基金
浙江省中医药科技计划项目(2021ZB342)。