慢病毒介导的稳定沉默nm23-H1基因的肺癌细胞株的建立及生物学行为改变  

Lentivirus-mediated Stable Silencing of nm23-H1 Gene in Lung Cancer Cells and the Influence on Biological Behavior

作　　者：罗猛[1,2,3] 朱大兴[1,2] 弓磊[1,2] 邱小明[1,2] 祖玲玲[1,2] 孙丽亚[1,2] 吴志浩[1,2] 周清华[1,2] 

机构地区：[1]天津市肺癌转移与肿瘤微环境重点实验室天津市肺癌研究所 [2]天津医科大学总医院,天津300052 [3]贵州省人民医院胸外科,贵阳550002

出　　处：《中国肺癌杂志》2012年第3期139-145,共7页Chinese Journal of Lung Cancer

基　　金：国家自然科学基金重点项目(No.30430300);国家自然科学基金项目(No.30973384);国家"863"重大项目(No.2006AAOZA401);国家"973"重大项目(No.2010CB529405);中瑞国际合作项目(No.09ZCZDSF04100)资助~~

摘　　要：背景与目的 nm23-H1基因是重要的肿瘤转移抑制基因。前期研究发现利用化学合成的小干扰RNA(small interfering RNA,siRNA)抑制nm23-H1基因的表达可明显增强肺癌细胞的侵袭力。为了进一步研究nm23-H1基因沉默后的分子生物学机制,本研究利用慢病毒介导的短发夹RNA(short hairpin RNA,shRNA)建立nm23-H1基因稳定沉默的肺癌细胞株。方法将表达特异性抑制nm23-H1基因shRNA的慢病毒转染人大细胞肺癌细胞株NL9980和肺腺癌细胞株A549,通过嘌呤霉素筛选出稳定转染细胞株。逆转录PCR、定量PCR及Western blot法检测nm23-H1基因表达,并通过shRNA抵抗的nm23-H1基因重组质粒转染拯救实验验证,侵袭小室实验检测侵袭力改变。结果逆转录PCR、定量PCR和Western blot法检测稳定转染细胞株NL9980-99和A549-99中nm23-H1基因在mRNA和蛋白水平表达均明显降低;shRNA抵抗的nm23-H1基因重组质粒转染拯救实验重现nm23-H1的正常表达;侵袭小室实验显示NL9980-99和A549-99细胞侵袭力明显增强。结论成功建立nm23-H1基因稳定沉默的人大细胞肺癌细胞株NL9980-99和人肺腺癌细胞株A549-99,nm23-H1基因沉默后使NL9980-99和A549-99细胞的侵袭力明显增强。Background and objective The nm23-H1 gene is an important tumor metastatic suppressor gene.Our previous study showed that the downregulation of nm23-H1 gene expression using small interfering RNA（siRNA）in NL9980 lung cancer cells greatly enhanced their invasiveness.To further explore the molecular mechanisms after nm23-H1 gene knock-down,we established transgene NL9980 and A549 lung cancer cell lines with stable nm23-H1 gene silencing through the lentivirus-mediated short hairpin RNA（shRNA）method. Methods The human large cell lung cancer NL9980 and human lung adenocarcinoma A549 cells were transfected with shRNA lentiviral particles specific for the nm23-H1 gene,and were then selected through puromycin.Puromycin-resistant clones were generated and screened using reverse transcription polymerase chain reaction（RT-PCR）,quantitative real-time polymerase chain reaction（qPCR）,and Western blot analysis.shRNA rescue experiments were performed to restore the nm23-H1 gene expression in the shRNA-expressing cells.Invasiveness was deter-mined through a Boyden chamber assay.Results The puromycin-resistant clones（NL9980-99 and A549-99）showed very low levels of nm23-H1 mRNA and protein expression under RT-PCR,qPCR,and Western blot analysis.Meanwhile,the shRNA rescue experiment restored the nm23-H1 expression in the NL9980-99 and A549-99 cells detected by Western blot.Down-regulation of nm23-H1 gene expression enhanced the invasiveness of the NL9980-99 and A549-99 cells compared with thecontrols.Conclusion The lung cancer cell lines NL9980-99 and A549-99 with stable nm23-H1 gene silencing were success-fully established and their invasiveness was greatly increased after nm23-H1 gene knockdown.

关 键 词：肺肿瘤 慢病毒 RNA 基因 

分 类 号：R734.2[医药卫生—肿瘤]