Yan Wei Hua, Lin Ai Fen, Chang Chien Chung, Ferrone Soldano
Laboratory Center, Wenzhou Medical College Affiliated Taizhou Hospital, Linhai 317000, China.
Cell Res. 2005 Jul;15(7):523-31. doi: 10.1038/sj.cr.7290376.
The non-classical HLA class I antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells (DC). As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-AC) and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5' regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components (APM) as well as beta2 microglobulin (beta2-m) expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1A cells.
非经典的I类人类白细胞抗原HLA - G是一种免疫调节剂，可抑制T细胞、自然杀伤细胞（NK细胞）和树突状细胞（DC）的功能。因此，恶性细胞中HLA - G的表达可能为它们提供一种逃避免疫监视的机制。在黑色素瘤中，已在30%的手术切除病变中发现HLA - G抗原表达，但在不到1%的已建立细胞系中发现该表达。体内和体外HLA - G表达差异的一种可能机制是，HLA - G基因在体外黑色素瘤细胞中受到表观遗传抑制。为了验证这一假设，我们用DNA甲基转移酶抑制剂5 - 氮杂 - 2'-脱氧胞苷（5 - AC）处理HLA - G阴性的黑色素瘤细胞系OCM - 1A，并分析HLA - G表达是否可以恢复。我们的数据有力地表明，HLA - G由于起始密码子上游220 bp的5'调控区域内的CpG高甲基化而沉默。处理后，HLA - G mRNA表达显著增加。蛋白质印迹法和流式细胞术显示诱导了HLA - G蛋白。有趣的是，5 - AC处理的OCM - 1A细胞上的HLA - G细胞表面表达远低于HLA - G阳性的JEG - 3细胞，而观察到的总HLA - G量相似。该研究分析了差异的可能机制，如细胞冷处理、肽负载和抗原加工机制成分（APM）以及β2微球蛋白（β2 - m）表达。数据显示，APM成分钙网蛋白可能与OCM - 1A细胞上较低的HLA - G表面表达有关。综上所述，我们的结果表明DNA甲基化是体外黑色素瘤细胞中调节HLA - G抗原表达的重要表观遗传机制。此外，我们首次假设钙网蛋白的缺乏可能与5 - AC处理的OCM - 1A细胞上较低的HLA - G表面表达有关。
