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

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 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 determined 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. Downregulation of nm23-H1 gene expression enhanced the invasiveness of the NL9980-99 and A549-99 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.