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-145-5pinhibitor+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 andmiR-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 verifiedthe 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-145-5pinhibitor+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 over- expression 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 proliferation of colorectal cancer cells and the cytotoxic effect of NK cells.

河北省医学科学研究课题计划（No. 20211322）