shRNA稳定抑制XIAP和survivin表达对胰腺癌细胞生物学特征的影响

目的:探讨慢病毒载体介导的RNA干扰(RNAi)技术对胰腺癌细胞X-连锁凋亡抑制蛋白(XIAP)和生存素(survivin)的抑制作用及对胰腺癌细胞增殖、凋亡及化疗敏感性的影响。方法:应用pGCSIL-PUR和pGCSIL-NEO分别构建针对XIAP和survivin的shRNA慢病毒载体,转染胰腺癌细胞株SW1990和Panc-1,经嘌呤霉素和新霉素筛选并扩大培养得到稳定转染株;实时荧光定量PCR和Western blot检测胰腺癌细胞内XIAP和survivin的表达;MTT法检测细胞增殖和化疗敏感性;caspase-3/7活性测定和DAPI染色分析细胞凋亡情况。结果:筛选XIAP和survivin的有效干扰靶点后,获得XIAP和survivin表达稳定抑制的SW1990和Panc-1细胞株。MTT检测显示稳定抑制XIAP或survivin后,两种胰腺癌细胞增殖均明显减弱,且两者联合抑制后作用增强;虽然抑制XIAP或survivin后对两种胰腺癌细胞凋亡无明显影响,但能明显增加其对化疗药物(5-FU,吉西他滨)的敏感性,且两者联合抑制后作用明显增强。结论:慢病毒载体介导的靶向XIAP和survivin的RNAi可有效抑制XIAP和survivin的表达,降低胰腺癌细胞的增殖能力并增强其对化疗药物的敏感性,且两者联合具有协同作用。

Influence of stable inhibition of XIAP and survivin expression on biological characteristics of pancreatic cancer cells

Objective: To investigate the inhibitory effect of lentiviral vector-mediated RNA interference(RNAi) on XIAP(X-linked inhibitor of apoptosis protein) and survivin expression in human pancreatic cells and its effect on the proliferation,apoptosis and chemosensitivity of pancreatic cells. Methods: The pGCSIL-PUR and pGCSIL-NEO were used to construct the lentiviral vectors containing shRNA targeting XIAP and survivin gene,respectively,and the human pancreatic cancer SW1990 and Panc-1 cells were transfected with these lentiviruses.The cells were screened by puromycin and neomycin double selection and expanded to obtain the stably transfected cell lines.The XIAP and survivin expressions were determined by real-time PCR and Western blot,respectively.The proliferation and chemosensitivity of the cells were detected by MTT assay,and caspase-3/7 activity measurement and DAPI-staining were performed to assess cell apoptosis. Results: The stable SW1990 and Panc-1 cells with inhibition of XIAP and survivin expression were obtained after the effective interference targets of XIAP and survivin screening.MTT results showed that the proliferative abilities of both types of pancreatic cells significantly decreased after the suppression of XIAP or survivin expression,and the combination inhibition of the two genes exerted a strengthened effect.Although XIAP or survivin inhibition had no obvious effect on apoptosis of both types of pancreatic cells,they enhanced the sensitivities of both types of cells to the chemotherapy agents(5-FU and gemcitabine),and this effect was also strengthened by their combination. Conclusion: The lentiviral vector-mediated RNAi targeting XIAP and survivin can effectively inhibit XIAP and survivin expression,and also inhibit proliferation and enhance chemosensitivity of pancreatic cells.The combination inhibition of both genes can exert a synergistic effect.