Natural Killer Cells Inhibit Oxaliplatin-resistant Colorectal Cancer By Repressing WBSCR22 Via Upregulating MicroRNA-146b-5p

Objective: Natural killer cells(NK cells)have a good anti-cancer application prospects.In this experiment,we will establish an oxaliplatin-resistant colorectal cancer cell model to elucidate the effect of natural killer cells on cell viability,proliferation,migration,and invasion of oxaliplatin-resistant cells,thereby exploring co-culture with natural killer cells.The expression level of miR-146b-5p in colorectal cells was changed to elucidate the regulatory relationship between WBSCR22 and miR-146b-5p.Finally,miR-146b-5p was explored to inhibit oxaliplatin-resistant colorectal cancer cells in natural killer cells.The role of malignant progression.Methods: The colorectal cancer cells RKO and DLD1 were cultured in RPMI-1640 medium,and oxaliplatin solution was added to establish resistant cell lines.CCK-8 cell proliferation assay and colony formation assay were used to detect the in vitro proliferation activity and colony formation ability of OR-DLD1 and OR-RKO cells.The in vitro migration and invasion of OR-DLD1 and OR-RKO cells were detected by cell scratch repair experiments and transwell cell invasion assays.The tumor-bearing test was used to examine the effect of natural killer cells on the allogenetic tumorigenicity of OR-DLD1 and OR-RKO cells.The mRNA and protein expression of WBSCR22 and the mRNA expression of miR-146b-5p in OR-DLD1 and OR-RKO cells were detected by real-time fluorescent quantitative PCR and Western blot.Through the miRanda online target prediction site miRanda,a double fluorescent reporter gene plasmid with the 3'UTR sequence of the WBSCR22 gene was cloned,and the predicted miR-146b-5p site was mutated to obtain a mutant plasmid.A cell line that stably knocked down miR-146b-5p expression was established and co-cultured with natural killer cells or co-injected to explore OR-DLD1-anti-control,OR-DLD1-anti-miR-146b-5p,OR-RKO-In vivo and in vitro functional differences between anti-control and OR-RKO-anti-miR-146b-5p cell lines.Results:1 On the basis of DLD1 and RKO cells,a drug-resistant cell line was established and named OR-DLD1 and OR-RKO.Relative to DLD1 and RKO cells,OR-DLD1 and OR-RKO cells underwent oxaliplatin-induced cell proliferation activity.Both colony formation and colony formation were significantly enhanced.2 The in vitro proliferative activity,colony formation,migration and invasion of OR-DLD1 and OR-RKO cells that were co-cultured with natural killer cells were significantly decreased,and Osali was co-injected with natural killer cells.The in vivo tumorigenicity of platinum-resistant cell lines OR-DLD1 and OR-RKO was significantly decreased.3 Through real-time fluorescence quantitative PCR experiments,we found that WBSCR22 mRNA expression was significantly down-regulated in OR-DLD1 and OR-RKO cells co-cultured with NK cells relative to control cells not co-cultured with NK.Through Western blot experiments,we found that WBSCR22 protein expression was significantly down-regulated in OR-DLD1 and OR-RKO cells co-cultured with NK cells relative to control cells not co-cultured with NK.MiR-146b-5p expression was significantly up-regulated in cells co-cultured with NK cells relative to cells not co-cultured with NK cells.We extracted miRNAs from OR-DLD1 and OR-RKO tumor xenografts in BALB/c nude mice and found that they were co-injected with NK cells by real-time PCR assays compared to tumor cells that were not co-injected with NK cells.The miR-146b-5p expression was significantly up-regulated in the transplanted tumor cells.4 The double fluorescent reporter gene assay showed that miR-146b-5p could significantly inhibit the luciferase activity of the 3? UTR wild-type plasmid with WBSCR22 attached,but had no significant effect on the luciferase activity of the mutant plasmid.The luciferase activity of the 3'UTR wild-type plasmid with WBSCR22 attached to the NK cell co-cultured cells was significantly reduced,while the luciferase activity of the mutant plasmid was not significantly changed.5 After inhibiting the expression of miR-146b-5p,natural killer cells were markedly impaired in the inhibition of cell viability,proliferation,migration,and invasion of OR-DLD1 and OR-RKO cells.Conclusion:OR1 OR-DLD1 and OR-RKO cells are oxaliplatin-resistant drug-resistant cell lines.2 Natural killer cells can inhibit cell viability,proliferation,migration and invasion of oxaliplatin-resistant colorectal cancer cell lines in vitro and in vivo.The above experiments show that NK cells can inhibit the expression of WBSCR22 mRNA and protein by up-regulating the expression of miR-146b-5p.miR-146b-5p inhibits WBSCR22 expression by targeting the 3'UTR of WBSCR22.3 MiR-146b-5p is a key effector molecule that natural killer cells inhibit cell viability,proliferation,migration and invasion of oxaliplatin-resistant colon cancer cells.