title: [Establishment of a nasopharyngeal carcinoma cell line with stable nestin silencing by lentivirus-mediated RNA interference].
abstract: OBJECTIVE
  To establish a nasopharyngeal carcinoma (NPC) cell line with stable nestin gene silencing induced by short-hairpin RNA (shRNA) interference.
  
  
  METHODS
  The nestin mRNA levels in 8 NPC cell lines were detected by real-time PCR and immunofluorescence assay. The recombinant lentiviral shRNA expression plasmid targeting nestin was packaged into mature lentivirus by 293T cells, and the supernatant containing the virus was harvested, concentrated and titrated. The best target for RNA interference was selected by real-time PCR. 5-8F cells was then infected by the recombinant lentiviral vector, and the expression of nestin in the cells was detected by Western blotting and real-time PCR.
  
  
  RESULTS
  The 8 NPC cell lines showed different nestin expression levels, among which 5-8F cells had the highest nestin expression. The recombinant lentiviral vector was successfully constructed and verified by PCR and sequencing. Nestin mRNA and protein levels was significantly reduced in 5-8F cells infected with shRNA-nestin lentivirus as compared with the negative control and the blank control cells.
  
  
  CONCLUSIONS
  Using the recombinant lentiviral vector constructed, we have successfully established a 5-8F NPC cell line with stable nestin gene silencing.
authors.affiliation: Cancer Institute, Southern Medical University, State Department of Education and Guangdong Provincial Key Laboratory of Transcriptomics and Proteomics for Major Diseases, Guangzhou 510515, China. pdslichuan@163.com
authors.forename: Chuan
authors.initials: C
authors.lastname: Li
authors.forename: Ji
authors.initials: J
authors.lastname: Ma
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.forename: Liang
authors.initials: L
authors.lastname: An
mesh_terms: D002277:Carcinoma; D045744:Cell Line, Tumor; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics; D000077274:Nasopharyngeal Carcinoma; D009303:Nasopharyngeal Neoplasms / Q000235:genetics* / Q000473:pathology; D009419:Nerve Tissue Proteins / Q000235:genetics*; D064231:Nestin; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D007381:Intermediate Filament Proteins; C577484:NES protein, human; D009419:Nerve Tissue Proteins; D064231:Nestin; D012333:RNA, Messenger; D034741:RNA, Small Interfering