Circ_0085315通过miR-149-5p/IRGQ通路对Treg介导的结肠癌免疫逃逸的机制探讨  

Study on mechanism of Circ_0085315 on Treg mediated immune escape of colon cancer through miR-149-5p/IRGQ pathway

作　　者：廖汉琪 唐羿[1] 胡凯[1] 姜耕[1] LIAO Hanqi;TANG Yi;HU Kai;JIANG Geng(General Surgery Department of the Second Hospital of Jingzhou,Jingzhou 434000,China)

机构地区：[1]荆州市第二人民医院普外科,荆州434000

出　　处：《中国免疫学杂志》2025年第4期915-924,共10页Chinese Journal of Immunology

摘　　要：目的:探讨Circ_0085315通过miR-149-5p/免疫相关GTP酶Q(IRGQ)通路对Treg介导的结肠癌(CC)免疫逃逸的机制。方法:收集50例CC患者癌组织及正常组织,体外培养CC细胞系(SW480、HCT116、HT29、SW620、Lovo)和人正常结肠上皮细胞HCoEpiC,qRT-PCR检测组织和细胞中Circ_0085315表达水平。以CC细胞系SW480细胞为研究对象,通过生物信息学分析、双荧光素酶报告基因实验、RNA结合蛋白免疫沉淀(RIP)实验验证Circ_0085315、miR-149-5p、IRGQ三者间的关系,qRT-PCR检测细胞中Circ_0085315、miR-149-5p和IRGQ mRNA表达水平,CCK-8检测细胞增殖活性,流式细胞术检测细胞凋亡率,Western blot检测细胞中IRGQ、Ki-67、PCNA、Bcl-2、Bax、Cleaved caspase-3蛋白表达,ELISA检测CC细胞与Treg共培养体系中免疫相关细胞因子水平,流式细胞术检测CC细胞与Treg共培养体系中FOXP3^(+)CD25^(+)、FOXP3^(+)CD4^(+)、FOXP3^(+)CD8^(+)比例。通过小鼠成瘤实验验证Circ_0085315对CC肿瘤生长、miR-149-5p/IRGQ通路以及免疫相关细胞因子的影响。结果:Circ_0085315在CC组织和细胞系中过表达。经生物信息学分析、双荧光素酶报告基因实验、RIP实验验证,miR-149-5p可能是Circ_0085315的下游靶点,IRGQ可能是miR-149-5p的一个靶基因。沉默Circ_0085315可降低CC细胞中IRGQ mRNA和蛋白表达水平、提高miR-149-5p表达水平(P<0.05);降低CC细胞存活率并提高细胞凋亡率,同时降低Ki-67、PCNA、Bcl-2蛋白表达水平,提高Bax、Cleaved caspase-3蛋白表达水平(P<0.05);还可降低CC细胞与Treg共培养体系中IL-10、TGF-β含量及FOXP3^(+)CD25^(+)、FOXP3^(+)CD4^(+)比例,提高IL-2、IFN-γ含量及FOXP3^(+)CD8^(+)比例(P<0.05)。进一步研究结果显示,抑制miR-149-5p表达可提高IRGQ mRNA表达水平,并减弱沉默Circ_0085315对CC细胞增殖、凋亡以及免疫逃逸的影响。体内小鼠成瘤实验显示,沉默Circ_0085315后肿瘤中IRGQ mRNA表达水平以及肿瘤体积、重量降低,而miR-149-5p表达�Objective:To investigate the mechanism of Circ_0085315 on Treg mediated immune escape of colon cancer(CC)through miR-149-5p/immunity-related GTPase Q(IRGQ)pathway.Methods:Cancer and normal tissues of 50 CC patients were collected,CC cell lines(SW480,HCT116,HT29,SW620,Lovo)and human normal colon epithelial cells HCoEpiC were cultured in vitro,and the expression level of Circ_0085315 in tissues and cells was detected by qRT-PCR.CC cell line SW480 was used as the research object,bioinformatics analysis,double luciferase reporter gene experiment and RNA binding protein immunoprecipitation(RIP)experiment were applied to confirm the relationship between Circ_0085315,miR-149-5p and IRGQ,qRT-PCR was used to detect the expression levels of Circ_0085315,miR-149-5p and IRGQ mRNA in cells,CCK-8 was used to detect cell proliferation activity,flow cytometry was used to detect the apoptosis rate,Western blot was used to detect the expression of IRGQ,Ki-67,PCNA,Bcl-2,Bax,and Cleared caspase-3 proteins in cells,ELISA was used to detect the level of immune related cytokines in the co-culture system of CC cells and Treg,flow cytometry was used to detect the proportion of FOXP3^(+)CD25^(+),FOXP3^(+)CD4^(+),FOXP3^(+)CD8^(+)in the co culture system of CC cells and Treg.The mouse tumorigenesis experiment was used to verify the influences of Circ_0085315 on CC tumor growth,miR-149-5p/IRGQ pathway and immune related cytokines.Results:Circ_0085315 was overexpressed in CC tissues and cell lines.According to bioinformatics analysis,double luciferase reporter gene experiment and RIP experiment,miR-149-5p might be the sion levels of IRGQ mRNA and protein,and increased the expression level of miR-149-5p in CC cells(P<0.05);it decreased the survival rate of CC cells and increased the apoptosis rate,decreased the expression levels of Ki-67,PCNA,Bcl-2 proteins,while increased the expression levels of Bax and Cleaved caspase-3 proteins(P<0.05);it could also reduce the contents of IL-10 and TGF-β,and the proportions of FOXP3^(+)CD25^(+)and FOXP3^(

关 键 词：Circ_0085315 miR-149-5p 免疫相关GTP酶Q TREG 结肠癌 免疫逃逸 

分 类 号：R735.35[医药卫生—肿瘤]