Abstract
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 col-lected,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 re-search 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 de-tect 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 cul-ture 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 downstream target of Circ_0085315,and IRGQ might be a target gene of miR-149-5p.Silencing Circ_0085315 decreased the expres-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 in-creased 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+CD4+in the co-culture system of CC cells,and increase the contents of IL-2 and IFN-γ,and the proportion of FOXP3+CD8+(P<0.05).Further research results showed that inhibition of miR-149-5p expression could increase the expression level of IRGQ mRNA and weaken the influences of silencing Circ_0085315 on CC cell proliferation,apoptosis and immune escape.In vivo mouse tumorigenesis experiment showed that after silencing Circ_0085315,the expression level of IRGQ mRNA,tumor volume and weight were decreased,while the expression level of miR-149-5p was increased(P<0.05);in addition,the contents of IL-10 and TGF-β in peripheral blood serum were decreased,while the contents of IL-2 and IFN-γ were increased(P<0.05).Conclusion:Silence Circ_0085315 can not only inhibit CC cell proliferation and induce apoptosis,but also inhibit Treg mediated CC immune escape by regulating miR-149-5p/IRGQ pathway.关键词
Circ_0085315/miR-149-5p/免疫相关GTP酶Q/Treg/结肠癌/免疫逃逸Key words
Circ_0085315/miR-149-5p/Immunity-related GTPase Q/Treg/Colon cancer/Immune escape分类
医药卫生
