title: Construction of a Hep-2 cell line stably transfected with Livin shRNA.
abstract: OBJECTIVE
  The aim of this study was to construct a eukaryotic expression plasmid with a short hairpin RNA (shRNA) targeting Livin in order to obtain a stably transfected Hep-2 cell line with a reduced expression of Livin.
  
  
  METHODS
  The shRNA targeting Livin mRNA was designed, and a shRNA plasmid and a negative control plasmid were constructed. After amplification in E. coli, restriction endonuclease digestion and sequence confirmation, the plasmids were transfected into Hep-2 cells using Lipofectamine 2000. The stably transfected cell line was screened using G418, and inhibition of Livin mRNA and protein levels were detected using real-time PCR and western blotting, respectively.
  
  
  RESULTS
  pGenesil-Livin-shRNA eukaryotic expression plasmid was successfully constructed and identified by sequencing. Green fluorescent protein (GFP) expression was observed in Hep-2 cells transfected with shRNA plasmids by fluorescence microscopy. The expression levels of Livin mRNA and protein decreased significantly in Hep-2 cells transfected with the shRNA recombinant plasmid. The mRNA level was reduced by 47.17 %, and the protein level was reduced by 34.25 %.
  
  
  CONCLUSIONS
  The shRNA eukaryotic expression plasmid targeting Livin was successfully constructed, which could significantly inhibit the expression of Livin in laryngeal cancer Hep-2 cells. This provides a basis for future research on the function of Livin in Hep-2 cells, and gene therapy for laryngeal cancer.
authors.forename: S L
authors.initials: SL
authors.lastname: Wang
authors.forename: W T
authors.initials: WT
authors.lastname: Deng
authors.forename: G F
authors.initials: GF
authors.lastname: Wen
authors.forename: C W
authors.initials: CW
authors.lastname: Li
authors.forename: Y J
authors.initials: YJ
authors.lastname: Zeng
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004926:Escherichia coli; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D007202:Indicators and Reagents; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics* / Q000378:metabolism; D008055:Lipids; D008856:Microscopy, Fluorescence; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C419866:BIRC7 protein, human; D007202:Indicators and Reagents; D051034:Inhibitor of Apoptosis Proteins; D008055:Lipids; C086724:Lipofectamine; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins
keywords: RNA interference; apoptosis regulatory proteins; cell culture; livin gene.