CTEN Induces Epithelial-mesenchymal Transition And Metastasis In Colorectal Cancer And Its Molecular Mechanism

Background:Colorectal cancer is a kind of cancer that occurs in the colon or rectum and is one of the most common types of digestive tract malignant tumors.The incidence of colorectal cancer is increasing year by year,which seriously endangers the physical and mental health of human beings.Metastasis is the main cause of death in patients with malignant tumors.Tumor metastasis is a multistage,multi-step complex process.More and more evidences show that epithelial-mesenchymal transition(EMT)plays a key and complex role in the invasion and metastasis of tumors.CTEN is not expressed in normal tissues such as small intestine,colon,liver,spleen,etc.It is often highly expressed in other tumors except prostate cancer,and is considered as one of the new oncogenes involved in the development of cancer.Whether CTEN promotes the migration and invasion of colorectal cancer through epithelial-mesenchymal transformation deserves further study.Objective:To explore the role of CTEN in epithelial-mesenchymal transformation,migration and invasion of human colorectal cancer and its possible molecular mechanism.Method:We collected the resected specimens of colorectal cancer tissues,detected the expression of CTEN protein in colorectal cancer tissues and adjacent colorectal tissues by IHC,analyzed the relationship between the expression of CTEN protein and the clinicopathological characteristics of patients with colorectal cancer,and survival analysis.Then we detected the expression of CTEN by quantitative PCR and Western blotting in three colorectal cancer cell lines and one normal colorectal cell line.Then we selected LoVo cells from colorectal cancer and constructed LoVo-CTEN cell line with high CTEN expression and LoVo-pCMV cell line as control.The The expression of E-cadherin,N-cadherin,Vimentin and transcription factor Twist in stable transformed cell line LoVo-CTEN and LoVo-pCMV were detected by quantitative PCR and Western blotting.The scratch assay and Transwell assay were used to detect the migration and invasive ability of LoVo-CTEN and LoVo-pCMV.Then we overexpressed the transcription factor Twist in LoVo cell line of colorectal cancer.Quantitative PCR and Western blotting were used to detect the changes of epithelial markers E-cadherin and interstitial markers N-cadherin and Vimentin.Scratch test was used to detect the migration ability of LoVo cell line.Transwell test was used to detect the invasion energy of LoVo cell line.Finally,we used Lipofectamine 2000 to transfect Twist interfering plasmid siTwist into human colorectal cancer LoVo cells as interference group,the control plasmid siNC was transfected into LoVo cells as control group.After 36 hours of transfection,we detected the expression of Twist in the two groups by quantitative PCR;after transfected 48 hours,Western blotting was used to detect the expression of transcription factor Twist protein in two groups of cells.Then we used Lipofectamine 2000 to transfect CTEN high expression plasmid pcmv-CTEN into two groups.After 36 hours of transfection,we used quantitative PCR to detect the changes of CTEN and EMT marker expression in mRNA level;After 48 hours of transfection,we detected the changes of CTEN and EMT marker expression in protein level by Western blotting.Results:The positive rate of CTEN protein expression in colorectal cancer tissues was 55.8%,which was significantly higher than that in adjacent colorectal tissues,and the difference was statistically significant.Furthermore,the expression of CTEN protein was significantly correlated with tumor size(P=0.001),differentiation(P=0.017),invasion depth(P=0.013)and lymph node metastasis(P=0.004)in colorectal cancer patients.Kaplan-Meier prognostic analysis showed that the survival time of patients with high expression of CTEN protein was significantly lower than that of patients with low expression of CTEN protein,and the expression level of CTEN protein could be used as an independent factor to predict the prognosis of patients with colorectal cancer(P<0.05).In cell experiments,Western blotting and quantitative PCR results showed that compared with normal colorectal cell line NCM460,CTEN was highly expressed in SW620,SW480 and LoVo cells,and the expression of CTEN protein in SW620 cells was the highest,followed by that in LoVo cells and the lowest in SW480 cells.We constructed stable transfected cell line LoVo-CTEN which overexpressing CTEN and control cell line LoVo-pCMV.We found that the interstitial markers N-cadherin and Vimentin tables were also raised,while the expression of E-cadherin was significantly down-regulated,and the migration and invasion ability of LoVo cell lines stably over-expressing CTEN was significantly enhanced,and the expression of transcription factor Twist was also up-regulated.Then we transfected LoVo cells with Twist plasmid for high expression of transcription factor Twist.In order to demonstrate Twist's involvement in CTEN's regulation of EMT,migration and invasion of LoVo colorectal cancer cell line,we first knocked down the expression of Twist in LoVo cell line by interfering RNA,then transfected the control plasmid siNC into LoVo cell line as control group,then transfected the CTEN high expression plasmid pcmv-CTEN into two groups of cells by Lipofectamine 2000.The changes of CTEN and EMT markers were detected by Western blotting and quantitative PCR.The results showed that the expression of N-cadherin and Vimentin in the interference group was lower than that in the control group,while the expression of E-cadherin protein was higher than that in the control group,and the ability of cell migration and invasion in the interference group was significantly decreased.This suggests that CTEN may play a role in promoting epithelial-mesenchymal transformation,migration and invasion of human colorectal cancer LoVo cells through Twist.Conclusion:(1)In colorectal cancer tissues,the expression of CTEN was significantly higher than that in adjacent colorectal tissues.Furthermore,the expression was closely related to tumor size,differentiation,depth of invasion and lymph node metastasis.The median survival time of patients with positive CTEN expression was significantly lower than that of patients with negative CTEN expression.This indicates that the prognosis of patients is poor.(2)CTEN can promote epithelial mesenchymal transition,migration and invasion of colorectal cancer cells.(3)CTEN promotes epithelial-mesenchymal transition,migration and invasion of colorectal cancer cells by up-regulating the expression of transcription factor Twist.