Nazari Nazanin, Farjadian Shirin
Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran.
Iran J Immunol. 2016 Sep;13(3):178-85.
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.
To evaluate the effect of HLA-G downregulation on NK cell cytotoxicity in tumor cell lines.
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.
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).
As a clinical approach, HLA-G downregulation with shRNA may be effective in cancer therapy by improving immune cell activation.
HLA - G是一种非经典的HLA I类分子，当在肿瘤细胞中表达升高时，它是肿瘤逃避包括自然杀伤细胞（NK）和T细胞在内的免疫反应的主要因素之一。
评估下调HLA - G对肿瘤细胞系中NK细胞细胞毒性的影响。
用靶向第4外显子特定序列的shRNA.1或靶向第4和第6外显子的shRNA.2转染SKOV3细胞后，通过实时聚合酶链反应（PCR）和流式细胞术测量HLA - G的表达水平。用乳酸脱氢酶（LDH）释放试验测量NK - 92MI细胞对转染或未转染的靶细胞系的细胞毒性。将Jeg - 3细胞系用作阳性对照。
与相应的未转染细胞相比，用两种短发夹RNA（shRNA）转染后，两种细胞系中膜结合的HLA - G表达水平均显著降低（p<0.05）。在用两种shRNA转染后的前48小时内 Jeg - 3细胞比SKOV3细胞更容易被NK细胞裂解。与未转染细胞相比，转染shRNA.1的SKOV3细胞在转染后24小时被NK细胞裂解的程度明显更高（p = 0.043）。
作为一种临床方法，用shRNA下调HLA - G可能通过改善免疫细胞激活而在癌症治疗中有效。
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