Clinical Significance And Mechanism Of SPOCD1 Expression In Colorectal Cancer

Objective:By exploring the expression of SPOCD1 in human colorectal cancer and its molecular mechanism of regulating colorectal cancer metastasis,this study provides new ideas and targets for the treatment of CRC and further reveals the role and related mechanisms of SPOCD1 in cancer.Methods:(1)The expression level of SPOCD1 m RNA in colorectal cancer was analyzed by using the TCGA,GEO data and TIMER database.Using TCGA and GEO data,ROC curve was developed to evaluate the diagnostic efficacy of SPOCD1 in differentiating CRC from normal tissues.(2)The TCGA,GEPIA and Prognascan databases were used to analyze the relationship between SPOCD1 and CRC prognosis.A Cox regression analysis was utilized to identify independent risk factors affecting prognosis.(3)GO,KEGG pathway analysis,and GSEA were used to identify possible cellular mechanisms for the role of SPOCD1 in CRC.(4)The correlation between SPOCD1 and Slug was analyzed through the GEPIA and Linkedomics online databases.(5)CIBERSORT algorithm and spearman correlation analysis were used to analyze the relationship between SPOCD1 and tumor immune infiltrating cells.(6)Immunohistochemical staining was used to detect the expression of SPOCD1 protein in CRC tissues.And the correlation between SPOCD1 gene expression and clinicopathological characters of CRC patients was analyzed.(7)Western blot analysis was used to analyze the expression of SPOCD1 protein in normal colorectal cell lines NCM460 and colorectal cancer cell lines HCT116 and Lo Vo.The expression of SPOCD1 in HCT116 colorectal cancer cells was knocked down by small interfering RNA(si RNA),and the expression of Slug was detected by western blot.Results:(1)Pan-cancer analysis based on TIMER database showed that the m RNA expression level of SPOCD1 in several kinds of cancers was significantly higher than that in the corresponding normal tissues,including breast invasive carcinoma,cholangiocarcinoma,colon adenocarcinoma,etc.In addition,the results of TCGA and GEO data also showed that the expression of SPOCD1 m RNA was significantly increased in CRC.SPOCD1gene had a good ability in differentiating CRC from normal tissues.(2)Survival analysis showed that high expression of SPOCD1 was related to adverse survival outcomes.Multifactorial COX regression analysis further supported that high SPOCD1 expression was an independent risk factor associated with prognosis in patients with CRC.(3)GO enrichment analysis showed that SPOCD1 was associated with extracellular matrix organization,extracellular structure organization,and positive regulation of MAPK cascade,etc.KEGG enrichment analysis showed that SPOCD1 was associated with PI3K-Akt signaling pathway,cell adhesion molecules,focal adhesion,proteoglycans in cancer,protein digestion and absorption,neutrophil extracellular trap formation,ECM-receptor interaction,Wnt signaling pathway,malaria,etc.The GSEA showed that substantial gene sets were positively enriched in SPOCD1 high-expression group including Epithelial-mesenchymal transition(EMT),apoptosis,Notch signaling pathway,TGF-βsignaling pathway and PI3K-Akt signaling pathway.(4)The expression of SPOCD1 was positively correlated with Slug expression.(4)CIBERSORT results showed that the high expression of SPOCD1 was negatively correlated with the infiltration levels of resting dendritic cells,activated memory CD4~+T cells and resting mast cells in tumor tissues(P<0.05),while the infiltration levels of monocytes and M0 macrophages increased in tumor tissues.Correlation analysis showed that the expression of SPOCD1 was correlated with 9 types of tumor infiltrating immune cells,and the expression of SPOCD1 was positively correlated with the infiltration levels of neutrophils,M0 macrophages and activated mast cells(P<0.05),and was negatively correlated with M1 macrophages,CD8~+T cells,resting mast cells,resting dendritic cells,activated memory CD4~+T cells and activated NK cells(P<0.05).(5)Immunohistochemical staining confirmed that the expression of SPOCD1 protein in CRC was higher than that in paired normal colorectal tissue.The expression of SPOCD1protein was not correlated with age,sex,tumor size and depth of invasion but was correlated with lymph node metastasis and vascular invasion.(6)Compared with normal colorectal cells(NCM460),SPOCD1 protein expression was increased in colorectal cancer cells(HCT116,Lo Vo).Using small interfering RNA to knockdown SPOCD1 expression,knockdown group’s Slug protein expression was also decreased compared with the negative control group.Conclusions:The m RNA and protein expression levels of SPOCD1 in CRC were significantly higher than those in normal colorectal tissues,and were associated with lymph node metastasis and vascular invasion.SPOCD1 is expected to become a new marker for the diagnosis and prognosis of CRC SPOCD1 protein promotes the development of colorectal cancer by regulating the EMT pathway.