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)期的比例明显升高,差异有统计学意义(t=9.614,P<0.05),处于S期的细胞比例相对降低;let-7amimic组SCCHN细胞凋亡率明显高于其他干预组(t=11.257,t=8.617,t=9.627;P<0.05);SCCHN细胞转染48 h后,let-7amimic组CD4^(+)、CD4^(+)与CD8^(+)比值(CD4^(+)/CD8^(+))水平最高,与其他4组比较差异有统计学意义(tCD4^(+)=9.257,t=8.654,t=6.647,t=7.152;tCD4^(+)/CD8^(+)=4.251,t=4.652,t=5.921,t=4.275;P<0.05);let-7ainhibitor组CD4^(+)和CD4^(+)/CD8^(+)水平最低,与其他4组比较差异有统计学意义(tCD4^(+)=4.357,t=5.267,t=5.267,t=5.415;t_(CD4^(+)/CD8^(+))=4.675,t=4.592,t=4.627,t=4.159;P<0.05)。结论:let-7a过表达可能通过抑制PD-1及PD-1L信号通路而参与SCCHN细胞免疫逃逸,其机制可能与靶向调控SOCS1有关。
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 relative expression levels of SOCS1,PD-1 and PD-L1 in let-7a inhibitor group were the highest,and the differences of them among let-7a inhibitor group and other intervention groups were statistically significant(t=6.357,t=9.647,t=7.256,P<0.05),respectively.The results of rtqPCR showed that the relative expression of let-7a mRNA was the highest in let-7a mimic group after transfection of human SCCHN cells for 48 h,and the differences of that among let-7a mimic group and other intervention groups were statistically significant(t=10.254,t=7.452,t=6.328,P<0.05),and the relative expression of let-7a mRNA in let-7a inhibitor group was the lowest,and there was statistical significance at that among let-7a inhibitor group and other intervention groups(t=6.257,t=6.901,t=5.287,P<0.05),respectively.The results of flow cytometry showed that the proportion of SCCHN cells at G_(0)/G_(1)phase in let-7a mimic group significantly decreased than that of blank control group(t=5.286,P<0.05),and the proportion of cells at S phase in let-7a mimic group relatively increased,while the proportion of CCHN cells at G_(0)/G_(1)phase in let-7a inhibitor group significantly increased,the difference was statistical significance(t=9.614,P<0.05),and the proportion of cells at S phase relatively decreased,and the apoptosis rate of SCCHN cells in let-7a mimic group was significantly higher than that in other intervention groups(t=11.257,t=8.617,t=9.627,P<0.05),respectively.After 48 h transfection of SCCHN cells,CD4^(+)and the ratio of CD4^(+)to CD8^(+)(CD4^(+)/CD8^(+))of let-7a mimic group had the highest level,and the differences of them between let-7a mimic group and other 4 groups were significant(tCD4^(+)=9.257,t=8.654,t=6.647,t=7.152,tCD4^(+)/CD8^(+)=4.251,t=4.652,t=5.921,t=4.275,P<0.05),respectively.At the same time,the CD4^(+),CD4^(+)/CD8^(+)levels of let-7a inhibitor group were the lowest,and the differences of them between let-7a inhibitor group and other 4 groups were significant(tCD4^(+)=4.357,t=5.267,t=5.267,t=5.415,tCD4^(+)/CD8^(+)=4.675,t=4.592,t=4.627,t=4.159,P<0.05),respectively.Conclusion:let-7a overexpression may be involved in the immune escape of SCCHN cell through inhibiting PD-1/PD-1L signaling pathway,which mechanism may be related to the targeted regulation of SOCS1.
作者
陈飞
李俊
杨宏山
余婷婷
张华萍
李凤阳
李璐
朱颖莺
CHEN Fei;LI Jun;YANG Hong-shan(Department of Oncology,The Central Hospital of Xiaogan,Xiaogan 432000,China)
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”。
作者简介
陈飞,男,(1985-),硕士,副主任医师,从事头颈及胸部肿瘤的临床研究工作;通信作者:朱颖莺,julianzyy@hotmail.com。
