The Effects And Mechanisms Of PCOLCE2 On Migration And Invasion Of Colorectal Cancer

Colorectal cancer is the third most common cancer in the world and one of the leading causes of cancer related deaths worldwide.Colorectal cancer was considered to be a disease that was mainly contributed by the developed countries,but in recent decades,the morbidity and mortality of colorectal cancer tends to rise in China,with the morbidity and mortality in urban area higher than that in the rural area.This trend can be explained by changes in diet and lifestyle westernization and the lack of early detection and early treatment for colorectal cancer.Due to the unnoticeable symptoms of colorectal cancer,only less than half of patients are diagnosed at the early stage.5-year relative survival rate of early patients is over 90%,while the 5-year relative survival rate of patients with distant metastasis is no more than 10%.Improving the prognosis and survival of patients with colorectal cancer is still an urgent task.Colorectal cancer is a heterogeneous disease associated with many genetic or somatic mutation,its development process involving complex abnormal gene expression and signaling pathways alteration.Along with the deepening understanding of the etiology and mechanism of colorectal cancer,researches in targeted therapy have made certain progress,but has not yet achieved satisfactory results.Further elucidating the pathogenesis of colorectal cancer and screening new biomarkers and therapeutic targets will provide new strategies for the targeted treatment of colorectal cancer.Epithelial-mesenchymal transition(EMT)is a common cellular biological process characterized by cells losing their epithelial properties,such as changes in cell polarity and loss of intercellular connection,while gaining mesenchymal properties,such as increased mobility.In colorectal cancer and other cancerous diseases,EMT plays an important role in tumor cell proliferation,invasion and metastasis,as well as in the acquisition of drug resistance.Procollagen C-proteinase enhancer protein2(PCOLCE2/PCPE2)is a kind of secretory glycoprotein in extracellular matrix,promoting the cleavage activity of C-proteinases of procollagen,such as BMP-1,by binding to COOH-terminal propeptide of procollagen,thereafter promoting the maturation and deposition of fibrocollagen,which functions crucially in the reorganization of extracellular matrix.Studies have found that the expression level of PCOLCE2 is predominantly related to the poor survival of thyroid cancer,head and neck squamous cell carcinoma,bladder cancer,gastric cancer and other tumors.However,the specific expression and function of PCOLCE2 in colorectal cancer has not been reported yet.In this study,the role and potential molecular mechanism of PCOLCE2 in colorectal cancer were studied by bioinformatics methods combined with in vitro cell experiments.Firstly,data of colorectal cancer in TCGA database were used to screen and analyze EMT-related genes,and a prognosis prediction model for colorectal cancer composed of four key genes was constructed.Besides,the prognostic model was verified in GEO dataset.Then,we further analyzed the relationship of PCOLCE2,one of the four key genes,with overall survival,clinicopathological parameters and immune infiltration level in colorectal cancer.Furthermore,the possible biological function of PCOLCE2 in colorectal cancer was analyzed through functional enrichment.Subsequently,PCOLCE2 expression levels in tumor tissues of colorectal cancer patients and colorectal cancer cell lines were investigated.In vitro experiments,it was found that overexpression of PCOLCE2 could enhance the ability of migration and invasion in colorectal cancer cells.As for the underlying mechanism,PCOLCE2 was found to regulate the expression of fatty acid binding protein-4(FABP4)and promote the EMT process of colorectal cancer cells.The purpose of this study is to provide a novel biomarker for colorectal cancer and new strategies for the targeted therapy.Part Ⅰ A novel prognostic risk score model for colorectal cancer based ontranscriptome sequencing of EMT-related genes Objective:To explore the relationship between key genes of epithelial-mesenchymal transformation(EMT)and prognosis of colorectal cancer,and to construct an individualized prognostic risk scoring model.Methods:Transcriptome sequencing data of Colorectal cancer and paracancerous normal tissue and clinical information were downloaded from TCGA and GEO databases,and EMT related gene set were downloaded from GSEA database.The differentially expressed genes(DEGs)between tumor and normal tissues were obtained by analyzing the sequencing data of TCGA,afterwards the differentially expressed EMT genes were obtained by intersection DEGs with EMT related gene set,and the prognostic genes were screened by univariate Cox analysis.LASSO Cox regression was used to construct the risk score model.Kaplan-Meier survival curve was used to analyze the survival differences between the high-and low-risk groups.The survival analysis of EMT risk score model was verified by GEO data cohort.Principal component analysis assessed the ability of genes in risk model to distinguish tumors and normal samples.The correlation between risk score model and prognosis was evaluated by univariate and multivariate Cox analysis.Subgroup survival analysis of risk score model was performed in different clinicopathological subgroups.Finally,clinical nomogram was constructed according to the risk score model and clinicopathological paremeters.Results:Four prognostic EMT genes,including PCOLCE2,OXTR,BDNF and SERPINE1,were screened from colorectal cancer samples in the TCGA database,based on which a risk score model was constructed.Survival analysis showed that the overall survival of the low-risk group was better than that of the high-risk group.ROC curve suggested the risk score model with good accuracy and AUC at 1,3 and 5 years were 0.8148,0.8627 and 0.8701,respectively.GEO data set verified the predictive ability of risk score model.Principal component analysis showed that the risk score model gene could distinguish tumor from normal tissue effectively.Univariate and multivariate Cox analysis showed that the risk score model was an independent prognostic factor.Subgroup analysis showed that patients in the high-risk group had poor prognosis in different groups of gender,age,T3-T4 tumor invasion,negative lymph node metastasis,no distant metastasis and early pathologic stage.Conclusion:The risk score model based on the key genes of EMT can accurately predict the overall survival of colorectal cancer patients,providing clues for the treatment and research of colorectal cancer,and the nomogram based on this model was helpful to evaluate the prognosis of patients.Part Ⅱ Prognostic value of PCOLCE2 in colorectal cancer Objective:To investigate the correlation between PCOLCE2 expression and prognosis in colorectal cancer patients.Methods:Colorectal cancer tissue and normal tissue samples were downloaded from TCGA and GEO databases to compare the expression level of PCOLCE2 in tumor and normal tissues.Kanplan-Meier survival curve was used to analyze the relationship between PCOLCE2 expression level and overall survival of patients with colorectal cancer.The relationship between PCOLCE2 expression level and age,sex and clinicopathological characteristics was analyzed.Biological functions related to PCOLCE2 were analyzed using GO functional annotation and KEGG pathway enrichment.The co-expressed genes of PCOLCE2 were analyzed by ULCAN database and DEGs in high and low expression groups of PCOLCE2.The relationship between PCOLCE2 and colorectal cancer immune invasion was analyzed by TIMER database.Results:Compared with normal tissues,PCOLCE2 expression level was lower in colorectal cancer tissues.However,survival analysis results showed that patients with higher PCOLCE2 expression level in colorectal cancer tissues had a shorter overall survival.The expression level of PCOLCE2 was correlated with pathological stage,tumor invasion degree,lymph node metastasis and distant metastasis.Functional analysis showed that PCOLCE2 was closely related to cell membrane function and extracellular matrix remodeling,and might be involved in calcium,c AMP and nerve signaling pathways.Coexpression gene analysis showed that PCOLCE2 was positively correlated with FABP4.Immune infiltration studies showed that PCOLCE2 was positively correlated with several kinds of immune infiltrating cells.Conclusion:Although the expression level of PCOLCE2 in colorectal cancer was lower than that in normal tissues,the increased expression level of PCOLCE2 in colorectal cancer was associated with poor prognosis of patients and was a potential prognostic marker of colorectal cancer.PCOLCE2 might be regulated by FABP4 and involved in the regulation of immune cell infiltration.Part Ⅲ Expression of PCOLCE2 in colorectal cancer Objective:To study the expression level of PCOLCE2 in colorectal cancer tissues and cells.Methods:The protein expression level of PCOLCE2 in tumor and paracancerous tissues of37 patients with colorectal cancer was detected by Western Blotting.The protein expression level of PCOLCE2 was detected in human colorectal cancer cell lines including HCT-8,SW620,HCT-15 and HCT-116 and normal colonic epithelial cell line NCM460.Results:Compared with normal tissues,PCOLCE2 expression level was lower in colorectal cancer tissues.Compared with NCM460 cells,the expression of PCOLCE2 was downregulated in HCT-8,SW620,HCT-15 and HCT-116 cells.Conclusion:PCOLCE2 expression level was lower in colon cancer tissues and cells than in normal tissues and cells,respectively.Part Ⅳ Effects of PCOLCE2 on migration and invasion of colorectal cancerand underlying molecular mechanism Objective:To investigate the biological functions and related mechanisms of PCOLCE2 in colorectal cancer cells.Methods:Colorectal cancer cell lines with PCOLCE2 silencing and overexpression were constructed by RNA interference and overexpression plasmid transfection in HCT-8cells with higher PCOLCE2 expression level and HCT-5 cells with lower PCOLCE2 expression level,respectively.Cell proliferation was detected by CCK8 and Ed U test.Cell migration and invasion were investigated by wound healing assay and Transwell assay.The protein expression level was detected by Western Blotting.Results:No significant difference was found in the proliferation of colorectal cancer cells with PCOLCE2 silencing and overexpression comparing to the control.The silencing of PCOLCE2 decreased the migration rate of colorectal cancer cells and the number of transmembrane cancer cells.The overexpression of PCOLCE2 increased the migration rate of colorectal cancer cells,and the number of transmembrane cancer cells.PCOLCE2 silencing promoted the expression of epithelial marker E-cadherin and inhibited the expression of mesenchymal marker Vimentin.Overexpression of PCOLCE2 inhibited the expression of E-cadherin and promoted the expression of Vimentin.PCOLCE2 silencing inhibited FABP4 expression while the overexpression of PCOLCE2 promoted the expression of FABP4.Conclusion:Overexpression of PCOLCE2 promoted migration and invasion of colorectal cancer cells in vitro,while silencing of PCOLCE2 inhibited migration and invasion of colorectal cancer cells in vitro.PCOLCE2 might regulate the EMT process of colorectal cancer cells by regulating FABP4 expression.