Natural Killer Cell Cytotoxicity Against SKOV3 after HLA-G Downregulation by shRNA.
BACKGROUND
HLA-G is a nonclassical HLA class I molecule which, when elevated in tumor cells, is one of the main factors involved in tumor evasion of immune responses including NK and T cells.


OBJECTIVE
To evaluate the effect of HLA-G downregulation on NK cell cytotoxicity in tumor cell lines.


METHODS
The expression level of HLA-G was measured by real-time PCR and flowcytometry after transfection of SKOV3 by shRNA.1, which targets specific sequences in exon 4, or shRNA.2, which targets both exons 4 and 6. NK-92MI cell cytotoxicity against transfected or untransfected target cell lines was measured with the lactate dehydrogenase (LDH) release assay. The Jeg-3 cell line was used as a positive control.


RESULTS
Membrane-bound HLA-G expression levels decreased significantly in both cell lines after transfection with both shRNAs compared to their corresponding untransfected cells (p<0.05). Jeg-3 cells were better lysed than SKOV3 cells by NK cells during the first 48 h after transfection with both shRNAs. Compared to untransfected cells, shRNA.1-transfected SKOV3 cells were significantly more lysed by NK cells 24 h post-transfection (p=0.043).


CONCLUSIONS
As a clinical approach, HLA-G downregulation with shRNA may be effective in cancer therapy by improving immune cell activation.
Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran.
Nazanin
N
Nazari
Shirin
S
Farjadian
D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic; D015536:Down-Regulation; D005260:Female; D059951:HLA-G Antigens / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000276:immunology*; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection; D019139:Tumor Escape
D059951:HLA-G Antigens; D034741:RNA, Small Interfering; D007770:L-Lactate Dehydrogenase