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). CONCLUSION: As a clinical approach, HLA-G downregulation with shRNA may be effective in cancer therapy by improving immune cell activation.
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). CONCLUSION: As a clinical approach, HLA-G downregulation with shRNA may be effective in cancer therapy by improving immune cell activation.