Mechanisms Of YAP Promoting Colon Cancer Cell Epithelial-mesenchymal Transition

Background: Colon cancer,one of the most common malignant tumors in the world and in China,ranks third.The incidence of colon cancer is hidden,and most patients with colon cancer have been accompanied by tumor metastasis at the first diagnosis.Although early surgery and radiotherapy and chemotherapy can treat and alleviate colon cancer to some extent,the therapeutic effect is not ideal for colon cancer patients with metastasis or recurrence.The pathogenesis of colon cancer is very complex,and the mechanism of colon cancer metastasis is more complex.The study on the mechanism of colon cancer metastasis will bring us opportunities for targeted treatment of colon cancer.YAP is the main effector downstream of Hippo pathway.It is highly expressed in colon cancer and is closely related to the degree of tumor metastasis.Epithelial-mesenchymal transformation makes tumor cells derived from epithelial cells lose cell polarity,obtain the characteristics of stromal cells,decrease adhesion ability,increase migration ability,and are more prone to tumor invasion and metastasis.Existing studies have shown that YAP is involved in the process of tumor epithelial-mesenchymal transformation and tumor invasion and metastasis,but its specific molecular mechanism is not clear.Thus,the study on the molecular mechanism of YAP promoting epithelial-mesenchymal transformation of colon cancer will help us to find a method for the treatment of tumor invasion and metastasis and provide new ideas for targeted therapy of colon cancer.Objective: The aim of this study was to explore the molecular mechanism of YAP promoting epithelial-mesenchymal transition in colon cancer has find a positive meaning for us to look for new targets for the clinical treatment of colon cancer.The effect of YAP expression on colon cancer EMT and the role of YAP in regulating Slug were studied by cell experiments,and the molecular mechanism of YAP regulating colon cancer invasion and metastasis will be further studied.Methods: Immunohistochemistry assays of YAP expression in clinically collected cancer tissues of patients with colon cancer,and the expression ofYAP in one kind of tissue cells and four kinds of colon cancer cells was detected by Western blot.The transfected cells were screened by limited dilution method,then the SW620 and HCT116 stable cell lines overexpressing YAP were constructed.The expression level of YAP protein and YAP m RNA were tested by Western blot and RT-q PCR respectively.The effects of overexpression of YAP and knockdown of YAP on the migration of different colon cancer cells were detected by Wound-Healing assay.The effects of overexpression of YAP and knockdown of YAP on the invasion of different colon cancer cells were detected by transwell invasion assay.Western blot was used to detect the expression of EMT-related molecules during overexpression of YAP and knockdown of YAP,and to clarify the effect of YAP on the regulation of EMT in colon cancer cells.Western blot is used to dectect the changes of EMT-related molecules in colon cancer cells transfected with overexpression YAP plasmid and Sh YAP plasmid.Western blot is used to detect the changes of E-cadherin expression by transiently transfected interfering Slug si RNA in stable SW620 cell line overexpressing YAP,and the expression changes of E-cadherin by transiently transfected overexpressing Slug plasmid in stable SW620 cell line knocking down YAP.Dual-Luciferase assay is used to detect up-regulation level of Slug transcriptional activity in stable SW620 cell line overexpressing YAP.The design α=0.05 is the significant level.The statistical methods of t-test and analysis of variance were used to analyze whether there was significant difference by SPSS17.0.Results: Immunohistochemistry results showed that compared with normal colonic mucosa,YAP expression was significantly increased in colon cancer tissues.Western blot results showed that compared with other tissue cells such as 293 T cells,the expression of YAP in colon cancer cells was also significantly increased.We successfully generated overexperss YAP in SW620 and HCT116 stably transfected cell lines with limited dilution method.Stable colon cancer cell lines were obtained after two weeks of puromycin screening.It can significantly increase the expression level of YAP protein and the level of m RNA.Cell wound-healing experiments showed that overexpression of YAPcan facilitate colon cancer cell migration,knockdown of YAP can inhibit colon cancer cell migration.Cell transwell invasion assay showed that overexpression of YAP can enhance colon cancer cell invasion,knockdown of YAP can weaken colon cancer cell invasion.In colon cancer cells stably overexpressing YAP,western blot showed that the EMT-related molecule E-cadherin expression was down-regulated and Slug expression was up-regulated.On the contrary,in colon cancer cell lines stably knocking down YAP,E-cadherin expression was up-regulated and Slug expression down-regulated.It was further confirmed that in colon cancer SW620 cells with transient overexpression of YAP plasmid,the expression level of Slug protein was significantly increased and that of E-cadherin was significantly decreased.In the colon cancer SW620 cells with transiently transfected with Sh YAP plasmid,the expression level of Slug protein was significantly down-regulated,and the expression level of E-cadherin protein was significantly increased.In addition,Western blot assay revealed that E-cadherin expression was up-regulated in transiently transfected si RNA targeting Slug in SW620 cells overexpressing YAP,E-cadherin expression was down-regulated in transiently transfected Slug plasmid in SW620 cell line knocking down YAP.We performed q PCR to detect Slug m RNA expression,and the result suggested that overexpress YAP increased Slug expression and YAP low expression decreased Slug expression.Dual-Luciferase assay revealed a significant increase in the luciferase activity of the Slug-Luc promoter in the SW620 cell line overexpressing YAP.Conclusion:(1)YAP can enhance the migration and invasion of colon cancer cells.(2)YAP can regulate colon cancer cells EMT.(3)YAP can promote EMT in colon cancer cells by inhibiting the expression of E-cadherin by activating Slug transcription.