Objective: To explore the effect and mechanism of c-Myc on regulating the expression of immune-related ligands in Y subtype small-cell lung cancer (SCLC) characterized by high expression of immune-related molecules. Methods: The Y subtype SCLC cell line H196 was randomly divided into the control group, c-Myc inhibitor 10058-F4 group, histone deacetylase 1 (HDAC1) inhibitor pyroxamide group, and 10058-F4 plus pyroxamide group. The co-culture system with NK-92MI cells was used to determine the effect of H196 cells on the function of natural killer (NK) cells. Western Blotting and co-immunoprecipitation assays were used to detect the effect of c-Myc on class Ⅰ HDAC, and flow cytometry was used to detect the regulatory effect of c-Mycon CD47, programmed cell death ligand 1 (PD-L1), and CD155, which are highly expressed immune checkpoints in Y subtype SCLC, and major histocompatibility complex classⅠ-related chains A and (MICA/B), which is a poorly expressed immune-activating ligand in SCLC, and the role of HDAC. Chromatin immunoprecipitation (ChIP) assay and real-time quantitative polymerase chain reaction (RT-qPCR) were used to determine the regulatory mechanism of c-Myc-HDAC1 on MICA/B expression. Results: Inhibition of c-Myc decreased the mortality of H196 cells in the co-culture system and down-regulated the expression of MICA/B. Compared with the NK+H196 group [(42.54±2.47)%], the proportion of cells killed by NK-92MI cells in the NK+H196+10058-F4 group was lower [(28.48±3.38)%, P＜0.001]. The mean fluorescence intensity (MFI) of MICA/B on the cells in the 10058-F4 group (36.40±0.82) was lower than that in the control group (91.23±8.60, P＜0.001). And c-Myc could bind to HDAC1, whose protein level was up-regulated by 10058-F4 while the mRNA level was not. Compared with the cells in the control group (90.10±4.91), the MFI of MICA/B on the cells in the pyroxamide group was significantly increased (145.70±5.86, P＜0.001), and the MFI of MICA/B on the cells in the 10058-F4+pyroxamide group (54.60±2.88) was significantly increased compared with the cells in the 10058-F4 group (35.97±1.60, P＜0.001). The percentage of MICA promoter gene fragments in the c-Myc antibody precipitation group (0.125±0.037) was significantly higher than that in the IgG group (0.000 8±0.000 3, P=0.004). MICB had a similar trend, suggesting that the c-Myc-HDAC1 complex could bind to the promoter region of MICA/B. The MFI of CD47 on the cells in the 10058-F4 group (60.07±0.21) was significantly lower than cells in the control group (70.27±1.37, P＜0.001), but the MFIs of PD-L1 (13.50±0.61) and CD155 (829.70±41.19) were significantly higher than those on the cells in the control group (9.23±0.94, P＜0.01; 496.00±4.36, P＜0.001, respectively). Conclusions: c-Myc may promote the expression of MICA/B and CD47 in Y subtype SCLC cells by binding and inhibiting HDAC1, while it may also be involved in inhibiting the expression of PD-L1 and CD155 in SCLC cells.
目的： 探讨以免疫相关分子高表达为特征的Y亚型小细胞肺癌（SCLC）中c-Myc对免疫相关配体表达的调控作用及机制。 方法： 以Y亚型SCLC细胞系H196为研究对象，分为对照组、c-Myc抑制剂10058-F4组、组蛋白去乙酰化酶1（HDAC1）抑制剂pyroxamide组以及10058-F4+pyroxamide组，采用与NK-92MI细胞共培养体系确定对自然杀伤（NK）细胞功能的影响，Western blot和免疫共沉淀实验检测c-Myc对Ⅰ类HDAC的作用，流式细胞术检测c-Myc对低表达的免疫激活性配体主要组织相容性复合体Ⅰ类链相关蛋白A和B（MICA/B）和Y亚型中高表达的免疫检查点分子CD47、细胞程序性死亡配体1（PD-L1）、CD155的调控作用及HDAC在其中的作用，染色质免疫沉淀检测和实时荧光定量聚合酶链反应确定c-Myc-HDAC1对MICA/B的调控机制。 结果： NK+H196+10058-F4组H196细胞死亡率［（28.48±3.38）%］低于NK+H196组［（42.54±2.47）%，P＜0.001］，10058-F4组H196细胞MICA/B的平均荧光强度（MFI）（36.40±0.82）低于对照组（91.23±8.60，P＜0.001）。c-Myc能够与HDAC1结合，10058-F4作用下HDAC1蛋白表达明显上调、mRNA表达水平无明显变化。pyroxamide组H196细胞表面MICA/B的MFI（145.70±5.86）高于对照组（90.10±4.91，P＜0.001），10058-F4+pyroxamide组H196细胞表面MICA/B的MFI（54.60±2.88）高于10058-F4组（35.97±1.60，P＜0.001）。机制研究发现，c-Myc抗体沉淀组MICA启动子基因片段百分比（0.125±0.037）高于IgG组（0.000 8±0.000 3，P=0.004），MICB有相似趋势，c-Myc-HDAC1复合物结合MICA/B启动子区。10058-F4组H196细胞CD47的MFI（60.07±0.21）低于对照组（70.27±1.37，P＜0.001），PD-L1的MFI（13.50±0.61）和CD155的MFI（829.70±41.19）高于对照组（9.23±0.94，P＜0.01；496.00±4.36，P＜0.001）。 结论： c-Myc可能通过结合和抑制HDAC1促进Y亚型SCLC细胞MICA/B和CD47的表达，同时还参与抑制SCLC细胞PD-L1和CD155的表达。.