抑制Rab25基因表达的人NSCLC厄洛替尼耐药细胞PC9/ER的建立  

Establishment of a stable erlotinib-resistant human non-small cell lung cancer cell line PC9/ER with lentivirus-mediated RNA interference for Rab25

作　　者：周建国[1] 罗清[1] 王菲[1] 王怡[1] 张钰[1] 吕水萍 柏玉举[1] 沈刚[1] 石磊[1] 马虎[1,2] 

机构地区：[1]遵义医学院附属医院肿瘤医院.遵义市肿瘤医院胸部肿瘤科肿瘤研究室.贵州省生物治疗人才基地,贵州遵义563000 [2]遵义医学院转化医学中心,贵州遵义563000

出　　处：《中华肿瘤防治杂志》2016年第21期1407-1413,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81360351;81660512);贵州省科技厅攻关项目(黔科字[2013]3003);遵义医学院博士启动基金(F-577);遵义医学院研究生社会实践项目(zy-yjs2015004);贵州省科技厅及遵义医学院;遵义市科技局联合基金重点项目(黔科合J字LKZ[2013]07号);贵州省高层次创新人才"千"层次人才基金

摘　　要：目的 Rab25在非小细胞肺癌(non-small cell lung cancer,NSCLC)等多种肿瘤中过度表达,提示其可能在NSCLC的发生发展及耐药形成中发挥重要作用。为进一步探讨Rab25的功能及其在NSCLC酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibitors,EGFR-TKIs)耐药形成中的作用,建立稳定慢病毒介导的shRNA靶向干扰Rab25基因人非小细胞肺癌厄洛替尼耐药细胞PC9/ER稳定株。方法实时定量PCR(real-time PCR,RT-PCR)检测PC9/ER及PC9细胞中Rab25基因mRNA的相对表达情况。筛选出Rab25基因的RNA干扰(RNA interference,RNAi)有效靶序列,合成靶序列的Oligo DNA并构建GV248-shRNA-Rab25慢病毒载体,酶切和测序鉴定正确后,经病毒包装,感染PC9/ER细胞,经嘌呤霉素筛选稳定表达细胞株。RT-PCR鉴定PC9/ER的表达,CCK-8检测对厄洛替尼的敏感性。结果 PC9/ER细胞中Rab25基因的mRNA表达水平显著高于PC9细胞。构建的重组慢病毒质粒经测序鉴定正确。RT-PCR证实,干扰Rab25后,PC9/ER细胞株中Rab25表达水平明显降低,抑制率为88.3%。通过传代10次后,PC9ER-Rab25i稳定细胞株中Rab25基因的mRNA表达水平显著低于阴性对照组,P<0.05。PC9ERRab25i稳定细胞株的IC_(50)为(2.133±0.222)μmol/L,显著低于阴性对照组的(6.375±0.799)μmol/L,P=0.007。结论成功构建了Rab25-shRNA慢病毒表达载体,建立了稳定抑制Rab25基因表达的人NSCLC厄洛替尼耐药细胞PC9/ER,初步验证Rab25基因能够改善肺癌EGFR-TKIs获得性耐药,为进一步研究Rab25在NSCLCEGFR-TKIs获得性耐药的机制及逆转其获得性耐药提供了可靠的细胞模型。OBJECTIVE The overexpression of Rab25 in many tumors including non-small cell lung cancer（NSCLC）reveals that Rab25 may contribute to the invasiveness and drug resistance of NSCLC.The objective of this study was to further study the biological function of Rab25 and its role in EGFR-TKIs resistance of NSCLC,we established a PC9/ER cell line for stable expression of shRNA targeting human Rab25 gene.METHODS A double-stranded shRNA targeting the Rab25 was designed,synthesized and inserted into a lentivirus vector（GV248）,and the insertion was identified by restriction endonuclease analysis and DNA sequencing.PC9/ER cells were then transfected with the packaged recombinant lentivirus,and resistant cell clones were selected with puromycin.The expression of Rab25 was examined using real-time PCR（RT-PCR）.After knocking-down Rab25 in PC9/ER with shRNA,the changes of inhibition rate and erlotinib IC50 were detected by CCK-8assay respectively.RESULTS The mRNA level of Rab25 was significantly higher in the PC9ER cells than in the PC9 cells.Sequencing results proved that recombinant lentivirus vector GV248-shRNA-Rab25 was constructed correctly.RT-PCR results suggested that the expression of Rab25 was significantly down-regulated in this infected PC9/ER cell line.The efficiency of siRNA interference of Rab25 could be up to 88.3%.The mRNA level of Rab25 was significantly lower in the stable PC9ER-Rab25 icells than that in the negative control cells.Erlotinib IC50 was much lower in the stable PC9ER-Rab25 icells than that in the negative control cells[（2.133±0.222）μmol/L vs（6.375±0.799）μmol/L,P=0.007].CONCLUSION The constructed and screened shRNA expression plasmid targeting at the Rab25 gene could suppress the Rab25 mRNA expression in PC9/ER cells significantly,which provides an experimental basis to study the effect of Rab25 gene silencing on the biological behavior of non-small cell lung cancer resistanced EGFR-TKIs by RNA interference.

关 键 词：RAB25 非小细胞肺癌 NSCLC 酪氨酸激酶抑制剂 耐药 慢病毒 稳定细胞株 

分 类 号：R734.2[医药卫生—肿瘤]