title: [Lentivirus-mediated stable silencing of nm23-H1 gene in lung cancer cells and the influence on biological behavior].
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Luo
authors.forename: Daxing
authors.initials: D
authors.lastname: Zhu
authors.forename: Lei
authors.initials: L
authors.lastname: Gong
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Qiu
authors.forename: Lingling
authors.initials: L
authors.lastname: Zu
authors.forename: Liya
authors.initials: L
authors.lastname: Sun
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Wu
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Zhou
mesh_terms: D045744:Cell Line, Tumor; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics* / Q000473:pathology; D054778:NM23 Nucleoside Diphosphate Kinases / Q000235:genetics* / Q000378:metabolism; D009361:Neoplasm Invasiveness; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism
chemical_list: D054778:NM23 Nucleoside Diphosphate Kinases; D034741:RNA, Small Interfering; C517938:NME1 protein, human