The Effect And Regulatory Mechanism Of Chronic Stress On The Epithelial-mesenchymal Transition And Cancer Stem Cells Process In Colorectal Cancer

Part1 The role of chronic stress in colon cancer epithelial-mesenchymal transition and tumor stem cellsObjective This study aims to clarify the role of chronic stress in epithelial-mesenchymal transition and cancer stem cells of colon cancer.Methods In order to explore the influence of chronic stress on the occurrence and development of CRC,BALB/c mice were used to construct chronic stress model,and then AOM/DSS was applied to induce the occurrence of CRC in chronic stress model mice and normal mice.Subsequently,immunohistochemistry(IHC)was used to evaluate the protein level of stemness-related markers(CD133,CD44),EMT-related markers(E-cadherin,Vimentin)and p53 in the tumor tissues of the two groups of mice.Enzyme-linked immunosorbent assay(ELISA)was used to detect the concentration of the main adrenal stress hormones(including cortisol,norepinephrine and epinephrine)in the peripheral blood of chronic stress-stressed mice.Epinephrine was used to stimulate the p53 wild-type cell lines HCT116 and Lovo,the expression of stemness-related markers,EMT-related markers and the p53 protein were detected.Subsequently,Subcutaneous xenograft tumor model was carried out in nude mice,western blotting and IHC experiments were used to detect the expression of stemness-related markers,EMT-related markers and the p53 protein.Results Chronic stress can promote the EMT and the stemness of CSCs in CRC.Long-term chronic stress can promote the occurrence of AOM/DSS-induced colon tumors.The immunohistochemical results show that the expression of p53 protein in the colon tissue of chronic stress model mice is significantly reduced,and the tumor stemness-related markers CD133 and CD44 are significantly increased.What’s more,the epithelioid marker E-cadherin was significantly reduced,and the mesenchymal marker vimentin was significantly increased too.Enzyme-linked immunosorbent assay(ELISA)was carried out to detect the contents of different adrenal stress hormones in the peripheral blood of the two groups of mice.The experimental results show that the concentrations of cortisol,norepinephrine and epinephrine in the peripheral blood of chronic stress model mice are all increased.In the p53 wild-type cell of colon cancer,after long-term treatment with low-dose EPI,the expression of p53 protein was decreased,the expression of mesenchymal marker protein(N-cadherin,Vimentin)was significantly increased,and the expression level of epithelial marker protein E-cadherin was significantly decreased.At the same time,the expression levels of CD133 and CD44,which are related to cell stemness,are significantly increased.In subcutaneous tumor formation experiments,the tumorigenesis ability of colon cancer cells was significantly enhanced after intraperitoneal injection of EPI,the expression of p53 protein was reduced,the expression of mesenchymal protein vimentin was significantly increased,and the expression level of epithelial characteristic protein E-cadherin was significantly reduced.The protein expression levels of CD133 and CD44,which are related to cell stemness,were significantly increased.Conclusion Long-term chronic stress can promote the epithelial-mesenchymal transition of colon cancer cells and the stemness of CSCs.Part 2 The mechanism of Chronic Stress Promoting the EMT and stemness of CSCs in Colon CancerObjective To further explore the molecular mechanism of chronic stress regulating the expression of p53 protein and promoting the EMT and stemness of CSCs in colon cancer.Methods Subcutaneous tumor formation experiments are applied to detect the effects of different adrenergic receptor agonist inhibitors on the growth of subcutaneous tumors in nude mice.Protein synthesis inhibitor cycloheximide(CHX)and proteasome inhibitor(MG132)are used to treat colon cancer cells,western blot and co-immunoprecipitation(co-IP)experimental are applied to explore the regulation of EPI on p53 protein.Western blot experiment and sphere culture experiment are applied to verify that EPI regulates the EMT and stemness of CRC by regulating the stability of P53 protein The E3 ligase TRIM2,which is regulated by EPI,was screened by transcriptome sequencing technology,and further verified by Western blot experiment and q RT-PCR experiment.HCT116 and Lovo cell were transfected with lentivirus-coated small interfering RNA or overexpression plasmids to knockdown or overexpress the expression of TRIM2,then,the expression of EMT related protein,stemness related protein and p53 protein were detected.CHX and MG132 were used to verify the regulation of TRIM2 on the ubiquitination and degradation of p53 protein.Co-immunoprecipitation(co-IP)and immunofluorescence co-localization experiments on TRIM2 protein and p53 protein were carried out to observe their mutual binding and co-localization in cell substructures in colon cancer cells.according to the structure of TRIM2 protein and p53 protein domains,plasmids containing tag and different structural domains were constructed to explore the direct binding domains of TRIM2 protein and p53 protein through co-IP experimental technology.Subsequently,western blot and q RT-PCR experiments were applied to further explore whether the changes in TRIM2 m RNA levels are regulated by p53,and then chromatin immunoprecipitation(Ch IP)and dual luciferase reporter gene were carried out to verify whether p53 can bind and regulate the TRIM2 promoter region.Finally,the western blot and sphere culture experiment were used to further verified whether TRIM2 can mediate the regulation of EPI on the expression of p53 protein,EMT and the stemness of CSCs.Results Chronic stress can significantly promote the occurrence and development of colon tumors.The release of epinephrine induced by chronic stress affects the EMT and the stemness of CSCs through the β2 adrenergic receptor(ADRB2).Epinephrine can promote the ubiquity of p53 protein.And overexpression of p53 can reverse the effect of epinephrine on EMT and stemness of CSCs in colon cancer cells.TRIM2,as an E3 ligase regulated by epinephrine,participates in the regulation of epinephrine on the ubiquitination of p53.TRIM2 directly binds to the p53 protein through its RING structure to promote the ubiquitination of p53.At the same time,as a transcription factor,p53 can feedback control the expression level of TRIM2 m RNA and inhibit the transcription of TRIM2 gene.Knockdown of TRIM2 protein can reverse the decrease expression of p53 protein caused by epinephrine in colon cancer cells,as well as the p53-mediated regulation of EMT and stemness of CSCs.Conclusion Long-term chronic stress can promote the occurrence and development of colon cancer.Under chronic stress,the secretion of epinephrine increases,which promotes the occurrence and development of colon tumors by acting on ADRB2 on the cell surface.In colon cancer cells,long-term low-dose epinephrine stimulation can enhance the transcriptional activity of TRIM2 and promote the expression of TRIM2 protein.The TRIM2 protein directly binds to the DNA binding domain(DBD)and transactivation domain(TAD)domains of the P53 protein through its RING domain,which promotes the ubiquitination of p53,increases the degradation of p53,and reduces the level of p53 protein in the cell.This leads to the EMT and promote the stemness of CSCs in colon cancer cells.In summary,our study explored the relationship between long-term chronic stress and the occurrence and development of colon cancer,and revealed that stress-induced epinephrine release promotes the occurrence and development of colon cancer through the TRIM2/P53 pathway.