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Archive > Volume 30 Issue 8 > 2023,30(8):681-688. DOI:10.3872/j.issn.1007-385X.2023.08.005 Prev Next
Basic Research
miR-145-5p affects the proliferation of colorectal cancer cells and cytotoxicity of NK cells by targeting regulation of RAD18 expression
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Abstract:
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.
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