title: RNA interfering targeting human leukocyte antigen-G enhanced immune surveillance mediated by the natural killer cells on hepatocellular carcinoma.
abstract: BACKGROUND
  Up-regulation of Human Leukocyte Antigen-G (HLA-G) expression is thought to contribute to the escape in immune surveillance by suppressing natural killer (NK) cell function. However, little is known about the expression of HLA-G in hepatocellular carcinoma (HCC) and its relationship to NK cell-mediated cytotoxicity. In this study, we aimed to investigate the expression of HLA-G in human HCC cell lines and determine whether its expression was related to inhibition of NK cell cytolysis.
  
  
  METHODS
  The levels of HLA-G gene expression in human HCC cell lines were assessed by indirect immunofluorescence analysis (IFA), Real time RT-PCR and Western Blot. Vectors containing small interfering RNA (siRNA) specifically targeting the HLA-G gene were constructed and applied to diminish HLA-G expression. The cells were examined by flow cytometry and fluorescent microscope 24 h after transfection as well as 2-3 weeks after G418 selection. The steady-state levels of HLA-G mRNA and protein were then checked by real time RT-PCR and Western Blot analysis, respectively. A nonradioactive cytotoxicity assay was used to evaluate the effect of HLA-G on NK-mediated cytotoxicity against the siRNA treated cells.
  
  
  RESULTS
  Both HLA-G mRNA and protein can be detected in human HCC cell lines. Levels of HLA-G mRNA and protein were diminished 88.73% and 75.91% respectively by targeting siRNA. In the stable HLA-G gene knock-down cell lines, a significant increase in NK cell-mediated lysis occurred.
  
  
  CONCLUSIONS
  Abnormal expression of HLA-G in HCC cell lines plays an important role in protecting against NK cell attack. The significant correlation between HLA-G expression and NK cell lysis implies that the abnormal expression of HLA-G might contribute to the mechanism of escape from host immune surveillance in HCC.
authors.affiliation: Department of Hepatobiliary Surgery, Zengcheng People,s Hospital (BoJi-Affiliated Hospital, Sun Yat-Sen University), Guangzhou, China.
authors.forename: Xian-Cheng
authors.initials: XC
authors.lastname: Zeng
authors.forename: Tong
authors.initials: T
authors.lastname: Zhang
authors.forename: De-Hao
authors.initials: DH
authors.lastname: Huang
authors.forename: Guo-Ying
authors.initials: GY
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.forename: Hua
authors.initials: H
authors.lastname: Li
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Tian-Ling
authors.initials: TL
authors.lastname: Fang
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhang
authors.forename: Gui-Hua
authors.initials: GH
authors.lastname: Chen
mesh_terms: D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000276:immunology*; D045744:Cell Line, Tumor; D003601:Cytotoxicity Tests, Immunologic; D017931:DNA Primers / Q000235:genetics; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D055785:Gene Knockdown Techniques; D059951:HLA-G Antigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007157:Immunologic Surveillance / Q000276:immunology*; D007694:Killer Cells, Natural / Q000276:immunology*; D008113:Liver Neoplasms / Q000276:immunology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D059951:HLA-G Antigens; D034741:RNA, Small Interfering
keywords: HLA-G; Hepatocellular carcinoma; Immune Surveillance; NK cytotoxicity; RNA Interfering