摘要
目的研究DNA依赖蛋白激酶催化亚基(DNA-PKcs)基因沉默后对Bel7402/5-氟尿嘧啶(5-Fu)肝癌耐药细胞的细胞周期及机制的影响,探讨DNA-PKcs影响化疗敏感性的相关机制。方法采用MTT法检测Bel7402/5-Fu细胞转染siDNA-PKcs后,对5-Fu、阿霉素(ADM)的敏感性;采用阳离子脂质体法将siDNA-PKcs寡核苷酸片段转染Bel7402/5-Fu细胞;流式细胞术检测细胞周期;Western blot法检测细胞P21、细胞周期蛋白B1(cyclin B1)、细胞分裂周期蛋白2(CDC2)的蛋白表达情况;实时定量PCR检测细胞毛细血管扩张共济失调突变基因(ATM)、p53的mRNA水平,Western blot法检测细胞ATM、P53蛋白水平。结果敲低细胞DNA-PKcs水平后,siDNA-PKcs组5-Fu、ADM的半数抑制浓度(IC50)明显降低,S期细胞减少、G2/M期细胞增多,细胞周期抑制因子P21蛋白水平增加,cyclin B1、CDC2蛋白水平降低,ATM、p53的mRNA及蛋白水平增加。结论敲低DNA-PKcs水平,增加P21蛋白水平,同时抑制cyclin B1、CDC2蛋白水平,诱导Bel7402/5-Fu细胞G2/M期阻滞,其机制可能与ATM-p53信号途径有关。
Objective To study the effect of the knock-down of the DNA-dependent protein kinase catalytic subunit（ DNA-PKcs） on the cell cycle of the multidrug-resistant（ MDR） Bel7402/5-Fu hepatocellular carcinoma cells and its MDR mechanism. Methods After cationic liposome-mediated siDNA-PKcs oligonucleotide transfection,the drug sensitivity of Bel7402/5-Fu cells to 5-fluorouracil（ 5-Fu） and adriamycin（ ADM） was determined by MTT assay; the cell cycle were detected by flow cytometry; meanwhile,the protein expressions of cel cycle-related proteins P21,cel cycle protein B1（ cyclin B1）,cel cycle division protein 2（ CDC2） were tested by Western blotting; the expressions of ataxia telangiectasia mutated（ ATM）and p53 at both mRNA and protein levels were detected by real-time PCR and Western blot analysis. Results The MTT results showed siDNA-PKcs increased the chemotherapeutic sensitivity of Bel7402/5-Fu cells to 5-Fu and ADM. The flow cytometric analysis showed siDNA-PKcs decreased the percentage of S-phase cells but increased the percentage of G2/M phase cells. Western blotting showed siDNA-PKcs increased the protein expression of P21 but decreased cyclin B1 and CDC2 proteins. In addition,siDNA-PKcs also increased the expressions of ATM and p53. Conclusion DNA-PKcs silencing increases P21 while decreases cyclin B1 and CDC2 expressions,and finally induces G2/M phase arrest in Bel7402/5-Fu cells,which may be related to ATM-p53 signaling pathway.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2017年第12期1647-1651,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
贵州省社法攻关项目[黔科合SY(2013)3008]
2016年硕士科研启动资金[F-829]
关键词
DNA依赖性蛋白激酶催化亚基
耐药肝癌细胞
细胞周期
机制
DNA-dependent protein kinase catalytic subunit
multidrug-resistant hepatocellular carcinoma cells
cell cyclemechanism
