let-7a靶向细胞因子信号传导抑制物1调控头颈部鳞状细胞癌免疫逃逸的作用机制研究  

Study on the effect and mechanism of let-7a targeted cytokine signaling inhibitor 1 in regulating the SCCHN immune escape

作　　者：陈飞[1] 李俊[1] 杨宏山[1] 余婷婷 张华萍[1] 李凤阳 李璐[2] 朱颖莺 CHEN Fei;LI Jun;YANG Hong-shan(Department of Oncology,The Central Hospital of Xiaogan,Xiaogan 432000,China)

机构地区：[1]孝感市中心医院肿瘤科,湖北孝感432000 [2]南方医科大学南方医院肿瘤科,广东广州510000 [3]华中科技大学同济医学院附属同济医院肿瘤科,湖北武汉430000

出　　处：《中国医学装备》2023年第5期22-28,共7页China Medical Equipment

基　　金：湖北省科技计划(2019CFB830)“let-7a靶向SOCS1调控PD-1/PD-L1信号通路在头颈部鳞状细胞癌免疫逃逸中的机制研究”。

摘　　要：目的:研究微小RNA亚型let-7a靶向细胞因子信号传导抑制物1(SOCS1)调控头颈部鳞状细胞癌(SCCHN)免疫逃逸的作用及机制。方法:常规培养人SCCHN细胞株舌鳞癌细胞(SCC25),采用双荧光素酶报告基因系统检测SOCS1是否为let-7a的作用靶点。将let-7a模拟物及阴性对照序列、抑制物及阴性对照分别转染人SCCHN细胞株SCC25,并将其分为空白对照组、let-7amimic组、mimic-NC组、let-7ainhibitor组和inhibitor-NC组。采用蛋白免疫印迹(Westernblot)法检测各组SOCS1和细胞程序性死亡-1(PD-1)及程序性死亡配体1(PD-L1)的蛋白相对表达量,实时荧光定量聚合酶链反应(rt-qPCR)检测各组let-7a、SOCS1与PD-1、PD-L1的mRNA相对表达量;转染48 h后,采用流式细胞术检测细胞周期、凋亡。T细胞亚群CD4^(+)、CD8^(+)分别与SCCHN细胞进行共培养,采用流式细胞术检测共培养组T细胞亚群CD4^(+)、CD8^(+)数目。结果:双荧光素酶报告基因实验分析结果显示,转染野生型载体的细胞荧光素酶活性均下调(F=26.566,P<0.05),提示let-7a可靶向结合SOCS13′UTR序列。Westernblot结果显示,空白对照组SOCS1蛋白相对表达量高于let-7amimic组;人SCCHN细胞转染48 h后,let-7amimic组SOCS1、PD-1及PD-L1蛋白相对表达量最低,与各干预组差异有统计学意义(t=6.427,t=7.102,t=5.287;P<0.05),let-7ainhibitor组SOCS1、PD-1及PD-L1蛋白相对表达量最高,与各干预组差异有统计学意义(t=6.357,t=9.647,t=7.256;P<0.05)。rt-qPCR结果显示,人SCCHN细胞转染48 h后,let-7amimic组let-7amRNA相对表达量最高,与各干预组差异具有统计学意义(t=10.254,t=7.452,t=6.328;P<0.05),let-7ainhibitor组let-7amRNA相对表达量最低,与各干预组差异有统计学意义(t=6.257;t=6.901,t=5.287;P<0.05)。流式细胞术检测结果显示,与空白对照组比较,let-7amimic组SCCHN细胞处于G0/G_(1)期的比例明显降低(t=5.286,P<0.05),处于S期的细胞比例相对增高,而let-7ainhibitor组CCHN细胞处于G0/G_(1)�Objective:To investigate the effect and mechanism of let-7a targeting cytokine signaling inhibitor 1(SOCS1)of microRNA subtypes in regulating the immune escape of squamous cell carcinoma of the head and neck(SCCHN).Methods:Tongue squamous carcinoma cell strain(SCC25)of human SCCHN cell line was routinely cultured,and the dual luciferase reporter system was used to detect whether SOCS1 was the target point of let-7a.Human SCCHN cell line SCC25 was transfected with let-7a mimic(mimic)and negative control sequences of mimic(mimic control),inhibitor(inhibitor)and negative control of inhibitor(inhibitor control)respectively,and they were divided into blank control group,let-7a mimic group,mimic-NC group,let-7a inhibitor group and inhibitor-NC group.Protein relative expression levels of SOCS1,programmed death 1(PD-1)and programmed death ligand 1(PDL1)of each group were detected by Western blot,and real-time fluorescence quantitative PCR(rt-qPCR)was used to detect the relative expression levels of the mRNA of let-7a,SOCS1,PD-1 and PD-L1 in each group.At 48th h after transfection,the cell cycle and apoptosis were detected by flow cytometry.The CD4^(+)and CD8^(+)of T cell subsets were respectively co-cultured with SCCHN cells.Flow cytometry was used to detect the number of CD4^(+)and CD8^(+)of T cell subsets in the co-cultured group.Results:The results of gene assay of double luciferase reporter showed that the luciferase activity of cells with transfected wild-type vectors was down-regulated(F=26.566,P<0.05),which indicated that let-7a could be targeted to combine with SOCS13´UTR sequence.Western blot results showed that the relative expression of SOCS1 protein in blank control group was significantly higher than that in let-7a mimic group,and the relative expression levels of SOCS1,PD-1 and PD-L1 proteins of let-7a mimic group were the lowest after 48 h of transfection,and the differences among let-7a mimic groups and other intervention groups were statistically significant(t=6.427,t=7.102,t=5.287,P<0.05),and the relat

关 键 词：头颈部鳞状细胞癌(SCCHN) 免疫逃逸 细胞因子信号传导抑制物1(SOCS1) let-7a 靶向调控 

分 类 号：R816.1[医药卫生—放射医学]