Effects of Abnormal Expression of MDM2 on Proliferation,Invasion and Migration of Gastric Cancer Cells and Immune In-filtration of CD4+T and CD8+T Cells
Objective To investigate the expression of murine double minute 2(MDM2)in gastric cancer and its effects on the proliferation,invasion,migration and immune infiltration of CD4+T and CD8+T cells.Methods The gas-tric cancer chip data set GSE62254 was extracted from Gene Expression Omnibus(GEO),and the expression of MDM2 in gastric cancer was statistically analyzed.The effects of MDM2 expression on the prognosis of patients with gastric cancer was analyzed by Gene Expression Profiling Interactive Analysis(GEPIA).The infiltration of 23 kinds of immune cells in GSE62254 was investigated by single-sample gene-set enrichment analysis(ssGSEA).The expressions of MDM2,CD4+T and CD8+T cells in clinical samples were detected by immunohistochemistry.The cell experiments were divided into sh group(siRNA-MDM2 down-regulated MDM2 expression)and si-NC group(siRNA-MDM2-NC not down-regulated MDM2 expression),si group(siRNA-MDM2 depressed MDM2 expression)and si-NC group(siRNA-MDM2-NC not de-pressed MDM2 expression),pc group(pcDNA3.1-MDM2 overexpressed on MDM2)and pc-NC group(pcDNA3.1-MDM2-NC not overexpressed on MDM2),mimic group(MDM2 mimic up-regulated MDM2 expression)and mimic-NC group(MDM2-NC not up-regulated MDM2 expression).The animal experiments were divided into si group(down-regu-lated MDM2 expression)and si-NC group(not down-regulated MDM2 expression),mimic group(elevated MDM2 ex-pression)and mimic-NC group(not elevated MDM2 expression),with 5 mice in each.The proliferation,migration and invasion of cells in each group were detected by 5-ethynyl-2'-deoxyuridine(EdU)and Transwell chamber.The growth and metastasis ability of cells in vivo were detected by animal experiments;the expression of MDM2,proliferating cell nu-clear antigen(PCNA),E-cadherin and Vimentin in cells and tumor tissues of mice were detected by Western blot;and the in vitro killing ability of CD8+T cells in single cell suspension of mice spleen tissues was detected by cell counting kit 8(CCK-8).Results Compared with adjacent normal tissues,the expression of MDM2 in gastric cancer tissue significantly increased(P＜0.05);compared with high MDM2 expression patients,patients with low MDM2 expression had a signifi-cantly higher survival curve in overall survival(P＜0.05);compared with the MDM2 high expression group,the infiltra-tion of CD4+T and CD8+T cells in the MDM2 low expression group significantly increased(P＜0.05);the immunohisto-chemical results of clinical samples showed a negative correlation between the expression of MDM2 and the infiltration lev-els of CD4+T and CD8+T cells.The cell experiment results showed that compared with the si-NC group,the proportion of EDU positive cells,the number of cell invasion and migration,the expression of MDM2,PCNA and Vimentin in PAMC-82 cells in si group significantly decreased,while the expression of E-cadherin significantly increased(all P＜0.05);compared with the mimic-NC group,the proportion of EdU positive cells,the number of cell invasion and migra-tion,the expression of MDM2,PCNA and Vimentin in PAMC-82 cells in the mimic group significantly increased,while the expression of E-cadherin significantly decreased(all P＜0.05);animal experimental results showed that compared with the si-NC group,the mass of tumor tissue,the proportion of lesion metastasis,the expression of MDM2,PCNA and Vimentin in tumor tissues of mice in the si group significantly decreased,the expression of E-cadherin in tumor tis-sues,immunohistochemical scores of CD4+T and CD8+T significantly increased(all P＜0.05);compared with the mim-ic-NC group,the mass of tumor tissues,the proportion of lesion metastasis,the expression of MDM2,PCNA and Vim-entin in tumor tissue of mice in the mimic group significantly increased,the expression of E-cadherin in tumor tissue and immunohistochemical scores of CD4+T and CD8+T significantly decreased(all P＜0.05).The in vitro killing effect of CD8+T cells on PAMC-82 cells in mice spleen tissues showed that the survival rate of cells in the si group significantly decreased(P＜0.05);compared with the mimic-NC group,the survival rate of the mimic group significantly increased(P＜0.05);the results of enzyme-linked immunosorbent assay(ELISA)showed that compared with the si-NC group,the contents of interferon gamma(IFN-y)and tumor necrosis factor alpha(TNF-α)in cell supernatant in the si group signifi-cantly increased(all P＜0.05);compared with the mimic-NC group,the contents of IFN-y and TNF-α in cell supernatant in the mimic group significantly decreased(all P＜0.05).Conclusion In gastric cancer,the expression of MDM2 increa-ses,the highly expressed MDM2 can significantly promote cell proliferation,migration and invasion,enhance the ability of tumor cells to resist CD4+T,CD8+T cells and promote the progression of gastric cancer.
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