Résumé
@#[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。
Résumé
Objective The objective of this study was to investigate the relationship between the expression of RAD18 and radiation resistance in glioblastoma multiforme(GBM)and to provide a new therapeutic target for improving the radiation resistance of GBM.Methods Human glioma A172 cells were transfected into blank and RAD18-containing plasmid vector.The cell proliferation of two groups after the same dose radiation was detected by cloning assay.The mRNA expression of RAD18 in primary and recurrent GBM samples after close proximity treatment were detected by qRT-PCR.All data were analyzed statistically.Results The proliferation of GBM cells transfected with RAD18 plasmid was higher than that of cells transfected with blank plasmid after radiation therapy(P<0.001).The expression level of RAD18 mRNA in recurrent GBM was higher than that in the untreated radioactive granules primary GBM(P<0.01).Conclusion The resistance of recurrent GBM to radiotherapy may be associated with the overexpression of RAD18 protein.