中国肺癌杂志2012，Vol.15Issue(3)：139-145,7.DOI:10.3779/j.issn.1009-3419.2012.03.02
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
摘要
Abstract
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 knockdown, 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 AS49 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 determined through a Boyden chamber assay. Results The puromycin-resistant clones (NL9980-99 and A549-99) showed very low levels of nm23-Hl 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 AS49-99 cells detected by Western blot. Down-regulation of nm23-H1 gene expression enhanced the invasiveness of the NL9980-99 and AS49-99 cells compared with the controls. Conclusion The lung cancer cell lines NL9980-99 and A549-99 with stable nm23-H1 gene silencing were successfully established and their invasiveness was greatly increased after nm23-H1 gene knockdown.关键词
肺肿瘤/慢病毒/RNA/基因Key words
Lung neoplasms/ Lentivirus/ RNA/ Genes分类
医药卫生引用本文复制引用
罗猛,来大兴,弓磊,邱小明,祖玲玲,孙丽亚,吴志浩,周清华..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[J].中国肺癌杂志,2012,15(3):139-145,7.基金项目
本研究受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)和中瑞国际合作项目(No.09ZCZDSF04100)资助 （No.30430300）
