Rna Interfering Targeting Human Leukocyte Antigen-G Enhanced Immune Surveillance Mediated by the Natural Killer Cells on Hepatocellular Carcinoma

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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.

Materials and 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.

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