Liu Jin Hong, Bian Yong Mei, Xie Yi, Lu Dao Pei
Department of Hematology, The Fifth People's Hospital of Shanghai, Fudan University, Shanghai 200240, P.R. China.
Department of Pediatrics, Minhang District Maternal and Child Health Care Hospital of Shanghai, Shanghai 201102, P.R. China.
Mol Med Rep. 2015 Jul;12(1):1059-65. doi: 10.3892/mmr.2015.3526. Epub 2015 Mar 20.
The aim of the present study was to explore the effect of epigenetic modification of class II transactivator (CIITA) methylation on histocompatibility complex (MHC) class II expression and the immune evasion of leukemia HL-60 cells. HL-60 cells were treated with various concentrations of 5-aza-2'deoxycytidine (5-Aza-CdR) and 0.5 µmol/l suberoylanilide hydroxamic acid (SAHA) for 24 h and then stimulated by interferon γ (IFN-γ) for 48 h. The mRNA levels of MHC class I, II and co-stimulatory molecules were quantified by reverse transcription polymerase chain reaction (RT-PCR). The levels of CIITA protein were determined by western blot analysis, and the CpG island methylation ratios in the CIITA promoter IV (CIITApIV) were analyzed by bisulfite-sequencing PCR (BSP). MHC I as well as the co-stimulatory molecules CD40 and CD80 were significantly increased following treatment with 5-Aza-CdR + SAHA + IFN-γ (epigenetic groups) compared with those in the control group and IFN-γ group (P<0.05). The expression of MHC class II and CIITA was restored and increased in an 5-Aza-CdR concentration-dependent manner in the three epigenetic groups. The results of the BSP assay showed that the methylation rate of CIITApIV CpG sites decreased with the treatment of epigenetic modification and negatively correlated to the 5-Aza-CdR concentration. This demonstrated that the negative expression of CIITA protein was the key reason for the loss of MHC II expression in HL-60 cells. The results of the present study may help to illustrate the mechanism of immune evasion in HL-60 cells.
本研究的目的是探讨II类反式激活因子（CIITA）甲基化的表观遗传修饰对白血病HL-60细胞的组织相容性复合体（MHC）II类表达及免疫逃逸的影响。用不同浓度的5-氮杂-2'-脱氧胞苷（5-Aza-CdR）和0.5 μmol/l的辛二酰苯胺异羟肟酸（SAHA）处理HL-60细胞24小时，然后用干扰素γ（IFN-γ）刺激48小时。通过逆转录聚合酶链反应（RT-PCR）定量MHC I类、II类及共刺激分子的mRNA水平。通过蛋白质免疫印迹分析测定CIITA蛋白水平，并通过亚硫酸氢盐测序PCR（BSP）分析CIITA启动子IV（CIITApIV）中的CpG岛甲基化率。与对照组和IFN-γ组相比，5-Aza-CdR + SAHA + IFN-γ处理组（表观遗传组）的MHC I以及共刺激分子CD40和CD80显著增加（P<0.05）。在三个表观遗传组中，MHC II类和CIITA的表达以5-Aza-CdR浓度依赖性方式恢复并增加。BSP分析结果显示，CIITApIV CpG位点的甲基化率随着表观遗传修饰处理而降低，且与5-Aza-CdR浓度呈负相关。这表明CIITA蛋白的阴性表达是HL-60细胞中MHC II表达缺失的关键原因。本研究结果可能有助于阐明HL-60细胞免疫逃逸的机制。
