Lentivirus-mediated Stable Silencing of nm23-H1 Gene in Lung Cancer Cells and the Influence on Biological Behavior
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 molecul...
Main Authors: | , , , , , , , |
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Format: | Article |
Language: | zho |
Published: |
Chinese Anti-Cancer Association; Chinese Antituberculosis Association
2012-03-01
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Series: | Chinese Journal of Lung Cancer |
Subjects: | |
Online Access: | http://dx.doi.org/10.3779/j.issn.1009-3419.2012.03.02 |
Summary: | 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. |
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ISSN: | 1009-3419 1999-6187 |