The Effects And Mechanisms Of PrPc-STAT3 Pathway In Chemotherapy-induced Epithelial-Mesenchymal Transition In Colorectal Cancer

Background & Objective:Colorectal cancer is one of the most common cancer in China.Surgery is given priority to the treatment of colorectal cancer,and it is supplemented by the comprehensive treatment of chemotherapy and other support treatments.Adjuvant chemotherapy can reduce the activity of tumor cells and decrease the small metastases.Neoadjuvant chemotherapy can relieve tumor load and lower the clinical stage.Consequently,chemotherapy is the most main treatment of colorectal cancer besides surgery.Although chemotherapy can effectively kill the cancer cells,some studies had shown that chemotherapy could increase tumor cell malignant phenotype,and tumor cells has stronger invasive ability after chemotherapy.Epithelial-mesenchymal transition(EMT)is the process that the polarized epithelial cells transform into mesenchymal cells,which have activity ability and could move freely in cell matrix.Its important features are more than the loss of cell polarity and the acquisition of mesenchymal characteristics.It is confirmed that EMT exists in a variety of epithelial tumors.It has close relationship with local invasion and distant metastasis of tumor cells.This study verified the relationship between chemotherapy and the occurrence of EMT in colorectal cancer cells respectively by vivo and vitro experiments.We hope the results could provide support for further investigating the molecular mechanism of EMT in colorectal cancer.Cellular prion protein(PrPc)is a kind of normal form of prion which is discovered freshly and exits in intra-cell.Through our previous experiments,our colleagues found that PRNP(gene encoding PrPc)changed most significantly after EMT in colorectal cancer cells(21.4 times).This study tried to validate whether PrPc plays a key role in the process of EMT in colorectal cancer respectively through vivo and vitro experiments.STAT3-Snail is the downstream effectors of numerous cytokine receptors and growth factor receptors signal.Correlation studies show that the activation of STAT3-Snail pathway is concerned with the occurrence of EMT in tumor cells.This study validate whether STAT3-Snail plays a key role in the process of EMT in colorectal cancer through a series of experiments.Methods: 1.We collect the wax samples from colorectal patients who successively received enteroscope biopsy 、 neoadjuvant chemotherapy(FOLFOX)and operation.Immunohistochemistry was used to detect the occurrence of EMT and the expression of PrPc in samples.2.χ2 and t test were used to analysis the relation between the occurrence of EMT and clinicopathologic characteristics of patients.3.DLD-1 and HT29 were treated with chemotherapy drugs 5-FU or oxaliplatin to establish EMT model.4.Inverted phase contrast microscope、confoeal laser scanning microscopy、Western blot and transwell assay were used to disturb or promote the expression of PrPc and Snail the occurrence of EMT and the changes of STAT3 and transcription factors.5.Transfect pCDNA3.0-PRNP 、 PrPc siRNA 、 siSnail into DLD-1(no-load transfection as control)to disturb or promote the expression of PrPc and Snail.6.Pull-down technology(Shanghai ZhuoKang biological co.,LTD)was used to detect the protein that was combines with PrPc after EMT in colorectal cancer cells.7.Co-immunoprecipitation assay was used to detect to the combination between STAT3 and PrPc after EMT in colorectal cancer cells.8.Transfect the luciferase reporter plasmid for E-cadherin and occluding、estrogen synthetase enzyme aromatase into DLD-1 for detecting the activity changes of promoter.Results:1.The results of detecting the clinical tissue specimens from colorectal cancer cells after chemotherapy by Immunohistochemical technology showed that cancer cells experienced EMT and the expression of PrPc elevated significantly.2.Whether or not the colorectal cancer cells after neoadjuvant chemotherapy experienced EMT that had something to do with microvascular invasion of cancer tissue and tumor cell differentiation level(p < 0.05)and that had nothing to do with the patients’ age,sex and tumor subtype(p>0.05).3.The results in vitro showed that the morphology of colorectal cancer cells experienced mesenchymal transition;The expression of epithelial markers decreased and the expression of mesenchymal markers inceased significantly;Cytoskeleton F-actin rearranged;The invasion ability of cells strengthened.4.Interferenece with the expression of PrPc in tumor cells by transfecting PrPc siRNA could restrain the occurrence of EMT and the strengthened invasion ability of cells induced by 5-FU.5.Transfection pCDNA3.0-PRNP could directly induce EMT in colorectal cancer cells.6.The results of detecting DLD-1 which had experienced EMT by pull-down technology show that STAT3(92kDa)combined most closely with PrPc.7.The results of detecting DLD-1 which were treated with 5-FU for 12 hours by Co-immunoprecipitation technology show that PrPc started to combine with STAT3,and phosphorylation of active site Tyr705 and Ser72 strengthened significantly.8.After transfecting pCDNA3.0-PRNP into DLD-1,we found that Snail was induced to express,and AZD9150(inhibitor of PrPc-STAT3 pathway)could restrain the process.Meanwhile the other transcription factors had no great changes.9.The results of detecting the luciferase reporter plasmid show that PrPc could restrain the activity of above-mentioned promoter,and the intervention measure of AZD9150、siSnail could restrain this process.10.The restrain of the expression of PrPc in DLD-1 which were treated with 5-FU could make Snail in nucleus disappear completely.For the cells in control group,the overexpression of PrPc could promote Snail to transfer from cytolymph to nucleus.11.After transfecting pCDNA3.0-PRNP into DLD-1 for 12 hours,Snail starts to express and to experience phosphorylation.Conclusion:Chemotherapy drug could induce EMT in colorectal cancer cells.The process of EMT in colorectal cancer cells induced by chemotherapy drug rely on the function of PrPc.PrPc may play a key role through downstream STAT3 pathway in the process of EMT in colorectal cancer cells which is induced by chemotherapy drug.During the process of EMT in colorectal cancer cells,PrPc-STAT3 pathway regulates the phosphorylation of Snail to influence the expression of E-cadherin and occluding.