| Objective(s): The role of LSECtin and CXCL8 in the treatment of colon cancer liver metastasis,tumor immune microenvironment,and immune checkpoint inhibitors(ICIs)has been widely recognized,but the regulatory role of CXCL8 on LSECtin expression remains unclear.We want to clarify the mechanism by which CXCL8 regulates the expression of LSECtin,and the effects of CXCL8 on cell proliferation and invasion.Methods:1.Database analysis: Data were obtained from the Cancer Genome Atlas(TCGA)and Gene Expression Ommba(GEO)databases.Differences in the expression of LSECtin and CXCL8 in colon cancer and normal mucosa were analyzed using the TCGA database and verified by GES110225 analysis.2.Clinical samples were used to analyze the difference in the expression of LSECtin and CXCL8 in colon cancer and normal mucosa.2.The differences between LSECtin and CXCL8 expression and immune cell infiltration,KEGG pathway,GO item,matrix score,estimated immune score,tumor mutation load,mismatch repair gene,and immune checkpoint expression in colon cancer were analyzed by database.3.Spearman method was used to analyze the correlation between LSECtin and CXCL8 in TCGA database and clinical samples.4.The expression of CXCL8 in HCT116,HT29,SW480,SW620,and RKO cells of different colorectal cancer cells was screened by Real-time quantitative PCR(q PCR).Two colon cancer cell lines with the highest expression of CXCL8 were selected.5.Mechanism study.Cell lines were selected and cultured,and RNA interference technology was used to knock down two colon cancer cell lines SW480 and SW620 with high CXCL8 expression,so as to explore and analyze the main regulatory effects of CXCL8 on the phenotype of colon cancer cells.The effect of CXCL8 knockdown on the proliferation of colon cancer cells was determined by MTT and plate cloning assay.6.western blot(WB)was used to preliminatively explore and analyze the correlation between the expression changes of CXCL8 and LSECtin in colon cancer cells,and clarify the expression mechanism.Results:1.CXCL8 was highly expressed in SW480 and SW620 cells,and low expressed in HT29 cells.2.Knocking down CXCL8 can inhibit the proliferation and invasion of SW480 and SW620 cells.3.At the protein expression level,knockdown of CXCL8 in SW480 and SW620 cells resulted in decreased expression level of LSECtin.4.In colon cancer tissues,the expression of CXCL8 was significantly higher than that in normal mucosa(P=0.0091),and there was a statistically significant difference in the expression level of LSECtin between colon cancer tissues and normal tissues,and the expression of LSECtin was significantly lower than that in normal mucosa.Expression of LSECtin and CXCL8 was positively correlated with CD8(+)T lymphocyte,DC cell,macrophage and neutrophil infiltration,stroma score,estimated immune score,tumor mutation load,and immune checkpoint expression.Expression of LSECtin is closely related to cytokine-cytokine receptor interaction pathways and responses to chemokine functions,such as CXCL8/CXCR1/2.LSECtin and CXCL8 are positively correlated with the expression of colon cancer.Cell experiments have demonstrated that the activation of AKT signal is an important pathway for CXCL8 to up-regulate the expression of LSECtin and promote the proliferation and invasion of colon cancer.Conclusion(s):1.At the cellular level,knockdown of CXCL8 can inhibit the proliferation and invasion of SW480 and SW620 cells.2.At the molecular level,knockdown of CXCL8 modulates LSECtin immune checkpoint expression via AKT signaling and is associated with immune microenvironmental remodeling in colon cancer.3.At the tissue level,the expression level of CXCL8 in colon cancer tissues was higher than that in normal tissues.The expression level of LSECtin in colon cancer tissues was different from that in normal tissues.The results of this study can provide theoretical basis for elucidating the primary drug resistance mechanism of ICIs,and also provide new ideas for solving the primary drug resistance problem of ICIs in colon cancer. |
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