c-Myc通过组蛋白去乙酰化酶1调控Y亚型小细胞肺癌免疫相关配体  

The effect of c-Myc on regulating the immune-related ligands in Y subtype small cell lung cancer through histone deacetylase 1

作　　者：赵沛妍 孙小单[1] 李慧[2] 田琳 陆元花 程颖[2,3] Zhao Peiyan;Sun Xiaodan;Li Hui;Tian Lin;Lu Yuanhua;Cheng Ying(Postdoctoral Research Workstation,Jilin Cancer Hospital,Changchun 130012,China;Translational Oncology Research Lab,Jilin Cancer Hospital,Changchun 130012,China;Department of Thoracic Oncology,Jilin Cancer Hospital,Changchun 130012,China)

机构地区：[1]吉林省肿瘤医院博士后工作站,长春130012 [2]吉林省肿瘤医院肿瘤转化医学实验室,长春130012 [3]吉林省肿瘤医院胸部肿瘤内科,长春130012

出　　处：《中华肿瘤杂志》2024年第11期1009-1018,共10页Chinese Journal of Oncology

基　　金：长春市科技发展计划资助项目(21QC05);国家自然科学基金(82103343);吉林省科技创新基地(平台)建设项目(YDZJ202202CXJD009);吉林省基础研究专项(202002062JC)。

摘　　要：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(0.0008±0.0003,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。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 classⅠHDAC,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

关 键 词：小细胞肺癌 C-MYC 组蛋白去乙酰化酶 主要组织相容性复合体Ⅰ类链相关蛋白A和B CD47 

分 类 号：R734.2[医药卫生—肿瘤]