| Colon cancer is one of the most common human gastrointestinal malignancies,which is high morbidity and mortality in the world,and even show a trend of increasing year by year.With the extensive application of epigenetics in cancer research,it will further uncover the complex pathogenesis and progression mechanism of colon cancer,provide a new theoretical basis and approach for molecular therapy of cancer,and develop more effective treatment strategies for a certain disease.Objective:To explore the expression of COL18A1 in colon cancer cell lines and tissues,to analyze the association between COL18A1 gene and clinicopathological features and prognosis of colon cancer,and to further analyze the possible biological processes and functions of COL18A1 gene in colon cancer by using bioinformatics methods.Methods:(1)The mRNA and protein expression levels of COL18AL gene in which human colon cancer HCT-116,HT29,SW620 cell lines and human normal colon mucosa FHC cell lines were detected by quantitative Real-time PCR(qRT-PCR)and Western-blot.(2)Colon Cancer tissue samples and normal colon tissue samples above 5cm adjacent to the cancer were selected from the specimen library,in which located in the Affiliated Hospital of North Sichuan Medical college.The relative expression level of COL18A1 Mrna,which are from cancer tissues and adjacent normal tissues of colon cancer patients are deteted by quantitative Real-time PCR(qRT-PCR).The relationship between COL18A1 expression level and clinicopathological features was analyzed by χ2 test,Cox survival regression risk model was used for univariate and multivariate analysis of the association between COL18A1 expression level and prognosis in colon cancer patients.And Kaplan-Meier method that has been by log-rank test was used to evaluate the difference in survival rate.When P-value was less than 0.05,the difference was considered statistically significant.(3)The co-expression gene information of COL18A1 was obtained from the Linkedomics database,and the biological processes and functions of related genes were determined through GO enrichment analysis and KEGG pathway analysis.The interaction network of proteins encoded by relevant genes of COL18A1 was analyzed using the STRING database,and finally,GO and KEGG mapping was performed using R language software.Results:1.The quantitative real-time PCR results showed that the expression of COL18A1 in colon cancer cell lines HCT-116,HT29 and SW620 in vitro was up-regulated compared with the normal colon mucosal epithelial cell lines FHC,and was significantly higher than that in the control group(P<0.05).2.Western Blot results showed that the protein expression of COL18A1 in cultured colon cancer cell lines HCT-116,HT29 and SW620 in vitro was significantly higher than that in the control group,and the difference was statistically significant.3.The highest COL18A1 relative express which is of 78 colon cancer tissues is 4.96,and rhelowest is 1.02,(high expression group:2.72±0.88,low expression group:1.62±0.53),69/78(88.46%)of patients with carcinoma of relative expression is higher than normal tissue adjacent to carcinoma,the t test analysis of COL 18A1 average expression level in cancerous tissue is higher than normal tissue adjacent to carcinoma,differences statistically significant.(P<0.001);4.The correlation between the high and low expression groups of COL18A1 gene and clinicopathological characteristics showed that:COL18A1 high expression level and age(χ2=0.149,P>0.05),gender(χ2=0.228,P>0.05),pathology types(χ2=0.609,P>0.05),tumor size(χ2=2.222,P>0.05),tumor location(χ2=3.087,P>0.05)has not significant correlation,Was significantly correlated with lymph node metastasis(χ2=4.743,P<0.05),and depth of invasion(χ2=9.447,P<0.05).5.Cox univariate analysis showed that the pathological type,lymph node metastasis,depth of invasion and expression level of COL18A1(P<0.05)were all relevant factors for the prognosis of colon cancer patients,Kaplan-Meier analysis showed that patients with high expression of COL18A1 had a lower overall survival rate.High expression of COL18A1,and lymph node metastasis(P<0.05)could be used as independent factors to predict low survival rate in patients with colon cancer.6.Bioinformatics analysis results showed that:GO enrichment analysis:COL18A1 closely involved in the cytoplasmic membrane,sexual extracellular matrix protein,extracellular matrix,cell surface components,with calcium ion,the structure of the extracellular matrix components,such as collagen molecules affect the proliferation and apoptosis of colon cancer cells,also through the cell adhesion,inflammation response,angiogenesis and signal transduction pathways regulating tumor occurred in the biological behavior of cancer cells.KEGG pathway enrichment analysis showed that COL18A1 co-expressed genes were mainly involved in cell adhesion factor,cytokine receptor interaction,RAP1 signaling pathway,PI3K-Akt pathway,focal adhesion,tumor signaling,ECM receptor interaction and other signaling pathways,and they were mostly related to tumor invasion,migration,proliferation and other behaviors.Protein interaction network showed that CTSL(cathepsin),COL6A2,ITGB1(integrin β1),MMP2(matrix metalloproteinase)and other proteins encoded by COL18A1 in the co-expression network were directly or indirectly interacted with the occurrence and development of colorectal cancer,gastric cancer,pancreatic cancer and other cancers.Conclusion:High expression of COL18A1 is associated with colorectal cancer cell lines and tissues,and is associated with depth of invasion and lymph node metastasis in patients about colorectal cancer.It may also have the potential to be a biomarker for diagnosis,treatment and prognosis evaluation of colon cancer. |
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