miR-145-5p通过靶向RAD18调控结直肠癌细胞的增殖与NK细胞的细胞毒作用  

miR-145-5p affects the proliferation of colorectal cancer cells and cytotoxicity of NK cells by targeting regulation of RAD18 expression

作　　者：李丽 姚红亮 LI Li;YAO Hongliang(Department of Surgery,Hengshui People's Hospital,Hengshui 053000,Hebei,China)

机构地区：[1]衡水市人民医院外科,河北衡水053000

出　　处：《中国肿瘤生物治疗杂志》2023年第8期681-688,共8页Chinese Journal of Cancer Biotherapy

基　　金：河北省医学科学研究课题计划(No.20211322)。

摘　　要：目的:探索RAD18影响结直肠癌细胞增殖及调节NK细胞对结直肠细胞的杀伤作用及其可能的机制。方法:采用生物信息学技术分析结直肠癌组织中RAD18和miR-145-5p的表达及两者之间的调控关系、分析RAD18富集通路。采用qPCR法验证RAD18和miR-145-5p在结直肠癌细胞中的表达,双荧光素酶报告基因实验验证miR-145-5p与RAD18的调控关系。按转染物的不同将SW480、HCT-15细胞分为将si-RAD18组、si-NC组,另向SW480细胞分别转染inhibitor-NC+si-NC、miR-145-5p inhibitor+si-NC或miR-145-5p inhibitor+si-RAD18,采用CCK-8法、克隆形成实验分别检测敲降miR-145-5p和/或RAD18对细胞增殖、克隆形成的影响;将各组细胞分别与经IL-2激活的NK92细胞共培养,采用乳酸脱氢酶释放法、ELISA和免疫荧光染色法分别检测NK细胞的细胞毒性、细胞因子分泌及细胞表面穿孔素和颗粒酶B表达的影响。结果:RAD18在结直肠癌组织和细胞中呈高表达(均P<0.01)。敲降RAD18可以抑制结直肠癌细胞增殖能力(P<0.05)和促进NK细胞活力、细胞毒性、IFN-γ、TNF-α、GM-CSF分泌及穿孔素和颗粒酶B的表达(均P<0.05)。双荧光素酶报告实验验证了RAD18-3’UTR与miR-145-5p的结合关系,miR-145-5p在结直肠癌组织和细胞中低表达(P<0.05或P<0.01)。miR-145-5p可以靶向下调RAD18的表达(P<0.05),过表达RAD18可以逆转miR-145-5p过表达对NK细胞杀伤效应的促进作用(均P<0.05)。结论:miR-145-5p可靶向下调RAD18的表达,miR-145-5p/RAD18轴能够影响结直肠癌细胞的增殖和NK细胞对其的细胞毒作用。Objective:To explore the possible mechanism of RAD18 affecting the proliferation of colorectal cancer cells and regulating the killing effect of NK cells on colorectal cells.Methods:Bioinformatics was used to analyze the expression of RAD18 and miR-145-5p in colorectal cancer tissues and the regulatory relationship between them,and to analyze the RAD18 enrichment pathway.The expression of RAD18 and miR-145-5p in colorectal cancer cells was verified by qPCR.Dual-luciferase reporter gene assay verified the regulatory relationship between miR-145-5p and RAD18.SW480 and HCT-15 cells were divided into si-RAD18 group and si-NC group according to the transfection;and SW480 cells were transfected inhibitor-NC+si-NC,miR-145-5p inhibitor+si-NC or miR-145-5p inhibitor+si-RAD18,respectively.The effects of knocking down miR-145-5p and/or RAD18 on cell proliferation and clonalization were detected by CCK-8 method and cloning formation experiments,respectively.The cells of each group were co-cultured with IL-2-activated NK92 cells,and the cytotoxicity,cytokine secretion,cell surface perforin and granzyme B expression of NK cells were detected by lactate dehydrogenase release assay,ELISA and immunofluorescence staining,respectively.Results:RAD18 was significantly over-expressed in colorectal cancer tissues and cells(all P<0.01).Silencing RAD18 can inhibit proliferation of colorectal cancer cells(P<0.05),promote NK cell viability,cytotoxicity,secretion of IFN-γ,TNF-α,GM-CSF and expression of perforin and granulozyme B(all P<0.05).In addition,dual-luciferase reporter assay verified the binding relationship between RAD18-3'UTR and miR-145-5p,which is underexpressed in colorectal cancer tissues and cells(P<0.05 or P<0.01).miR-145-5p can down-regulate the expression of RAD18 by targeting(P<0.05),and over-expression of RAD18 can reverse the promotion effect of miR-145-5p overexpression on NK cell killing(all P<0.05).Conclusion:miR-145-5p can target down-regulation of RAD18 expression,and the miR-145-5p/RAD18 axis can affect the prol

关 键 词：结直肠癌 miR-145-5p RAD18 NK细胞 SW480细胞 HCT-15细胞 

分 类 号：R735.3[医药卫生—肿瘤] R730.54[医药卫生—临床医学]