title: [Construction of A eukaryotic expression vector carrying the iNOS gene and its effect on A549 lung cancer cells].
abstract: OBJECTIVE
  The iNOS gene is associated with NO-mediated antitumor effects. The aims of this study are to construct a eukaryotic expression plasmid that carries the iNOS gene and to detect the expression levels and antitumor effects of the iNOS gene on A549 lung cancer cells.
  
  
  METHODS
  A DNA fragment of the human iNOS coding sequence was amplified using reverse transcription polymerase chain reaction (RT-PCR). The DNA fragment was subsequently cloned into the multiple cloning sites of the eukaryotic expression vector pVAX. The recombinant plasmid was confirmed using restriction enzyme treatment, PCR, and sequencing and was then transfected into A549 lung cancer cells. The expression of the iNOS gene in the A549 lung cancer cells after transfection was verified by RT-PCR and Western blot analysis. The effects of iNOS on cell apoptosis, proliferation, and migration were identified by staining with Hoechst 3235, an MTT assay, and a scratch assay, respectively.
  
  
  RESULTS
  The results of the restriction enzyme digestion, PCR, and sequencing verified the successful construction of the eukaryotic expression plasmid pVAX-iNOS. The iNOS gene expression level was increased in the transfected A549 cells. Further experiments also showed increased cell apoptosis among the A549 lung cancer cells transfected with pVAX-iNOS. Meanwhile, the proliferation and migration of A549 cells were significantly inhibited by the enhanced iNOS gene expression.
  
  
  CONCLUSIONS
  The recombinant eukaryotic expression vector pVAX-iNOS was successfully constructed and transfected into A549 cells. The enhanced iNOS gene expression significantly promoted cell apoptosis, whereas the proliferation and migration of A549 cells were inhibited. These findings contribute to the development of novel and effective gene therapies for lung cancer.
authors.affiliation: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China.
authors.forename: Sujuan
authors.initials: S
authors.lastname: Ye
authors.forename: Weihan
authors.initials: W
authors.lastname: Yang
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Wenjing
authors.initials: W
authors.lastname: Ou
authors.forename: Qingping
authors.initials: Q
authors.lastname: Ma
authors.forename: Wen
authors.initials: W
authors.lastname: Zhu
mesh_terms: D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D052247:Nitric Oxide Synthase Type II / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D052247:Nitric Oxide Synthase Type II