| Background and ObjectiveColorectal cancer is a common gastrointestinal tumor,and its incidence is increasing year by year and younger.According to the global cancer statistics in 2020,colorectal cancer ranks third in the incidence of all cancers and second in the mortality rate of all cancers,which seriously endangers human health.And about 20%-40% of patients have been combined with distant metastasis when colorectal cancer is diagnosed.Metastasis is the main cause of death in colorectal cancer patients.Therefore,we will explore the molecular mechanisms related to colorectal cancer metastasis and look for new colorectal cancer metastases.Molecular markers are a hot topic in current tumor research.Tumor microenvironment(TME)refers to the special environment for tumor cell growth formed by the interaction of tumor cells and extracellular matrix during tumor cell growth.TME is the place where tumor cells depend for survival.Tumor cells,mesenchymal cells and various cytokines released by them and extracellular matrix form a network that regulates tumor angiogenesis.The activation of vascular endothelial cells is the key to tumor angiogenesis.Because angiogenesis depends on the activation,migration of endothelial cells and the formation of vascular lumen.The angiogenesis switch is activated and new blood vessels are continuously generated,which not only provides nutrients for tumor growth,but also lays the foundation for tumor metastasis.Neovascularization in TME is an important basis for tumor cell proliferation and invasion of surrounding tissues,especially for distant metastasis.Formin-like 2(FMNL2)gene is one of the members of the Formins family.It is an important actin nucleation factor that can promote actin polymerization.According to reports in the literature: FMNL2 is dysregulated in a variety of cancers such as colorectal cancer and melanoma,and is involved in the invasion and progression of cancer cells.But its potential interacting protein and its mechanism of action are still unclear.In previous work,we found that FMNL2 is up-regulated in highly metastatic colorectal cancer cell lines and tissues.Through the yeast two-hybrid technology,EGFL6 with strong binding specificity to FMNL2 was screened as the follow-up research object.Epidermal growth factor-like-domain 6(EGFL6)is a new member of the epidermal factor-like domain superfamily.EGFL6 is a secreted protein.The literature search results suggest that it may participate in the vascular microenvironment.Regulation.It has EGF domain and MAM domain,also known as MAEG.EGFL6 exists in two forms in the microenvironment: membrane-bound protein and extracellular matrix protein.EGFL6 contains 1 N-terminal signal peptide,4 half-EGF repeats,1 integrin binding sequence and 1 C-terminal MAM domain.The MAM domain exists in the extracellular region of many proteins with diverse functions and has cell adhesion.Potential.Wang X and other studies have found that compared with normal tissues,EGFL6 is not only highly expressed in breast cancer,lung cancer,meningioma,melanoma and ovarian cancer,but also highly expressed in the endothelial cells of lung cancer,meningiomas and ovarian cancer.Western blot results also showed that EGFL6 can activate ERK1/2 signaling pathway after acting on vascular endothelial cells.The ERK1/2 signaling pathway is an important pathway that regulates cell proliferation,apoptosis and differentiation in many tissues.The ERK1/2 signaling pathway can promote the proliferation of vascular endothelial cells and the formation of new blood vessels.After the formation of new blood vessels,it can provide more nutrients to the tumor,accelerate the growth of the tumor,and promote the metastasis of cancer cells.The purpose of this experiment is to clarify the regulatory relationship between FMNL2 and EGFL6 and the interaction between EGFL6 and CKAP4.To clarify the role and mechanism of FMNL2/EGFL6/CKAP4 in the angiogenesis and metastasis of colorectal cancer.Provide new gene targets for the clinical diagnosis and treatment,prognosis and drug screening of tumor metastasis.Methods1.Detect the expression of FMNL2 in NCM460 cell lines and colorectal cancer cell lines by western blot and real-time quantitative PCR(RT-q PCR)technology.Construct a stable cell line that overexpresses and interferes with FMNL2,and verifies FMNL2 by in vivo and in vitro experiments such as Transwell experiment,chicken embryo allantoic membrane experiment,tubule formation experiment,nude mouse subcutaneous tumor model,tail vein lung metastasis model,and cecal orthotopic implant liver metastasis model.Promoting angiogenesis and metastasis.2.Verify the interaction between FMNL2 and EGFL6 by immunofluorescence,immunoprecipitation Co-IP,and GST pull down experiments.3.Detect the expression of EGFL6 in NCM460 cell line and colorectal cancer cell line by western blot and RT-q PCR technology.ELISA and Western blot were used to detect the expression of EGFL6 in the supernatant of colorectal cancer cells after overexpression of FMNL2.The morphology of extracellular vesicles was observed by transmission electron microscope,and the extracellular vesicles were extracted and identified using their universal markers.Immunofluorescence locates extracellular vesicles in colorectal cancer cell lines.Construct a stable cell line that overexpresses and interferes with EGFL6,and verifies FMNL2 by in vivo and in vitro experiments such as chicken embryo allantoic membrane experiment,Transwell experiment,tubule formation experiment,nude mouse subcutaneous tumor model,tail vein lung metastasis model,and cecal orthotopic implant liver metastasis model.Regulate the angiogenesis and metastasis of colorectal cancer through EGFL6 paracrine.Western blot was used to detect the expression and correlation of FMNL2,EGFL6,and CD31 in colorectal cancer paired tissues.Immunohistochemistry was used to detect the expression of FMNL2,EGFL6,and CD31 in normal intestinal mucosal epithelial tissues and different colorectal cancer tissues.4.Immunofluorescence and immunoprecipitation Co-IP experiments verified the interaction between EGFL6 and CKAP4.Western blot detection of signal pathways.Results1.The results of Transwell experiment,chicken embryo allantoic membrane experiment and tubule formation experiment show that FMNL2 can promote the migration of HUVEC and angiogenesis,and the difference is statistically significant(P<0.05).The nude mouse subcutaneous tumor model and the immunohistochemical results of subcutaneous tumor showed that FMNL2 can promote the formation of blood vessels in the body,and the difference was statistically significant(P<0.05).The experimental results of tail vein lung metastasis model and cecal orthotopic implant liver metastasis model showed that FMNL2 can promote the lung and liver metastasis of colorectal cancer,and the difference was statistically significant(P<0.05).2.The results of immunofluorescence and co-immunoprecipitation CO-IP show that EGFL6 can interact with FMNL2,and the GST-pull down result shows that EGFL6 can directly bind to the FMNL2-FH3(23-469AA)domain.3.The content of EGFL6 in the conditioned medium was detected by western blot and ELISA.The results showed that the content of EGFL6 in the supernatant of colorectal cancer cells overexpressing FMNL2 increased,and the difference was statistically significant(P<0.05).The extracellular vesicles were circular or elliptical when observed by transmission electron microscopy.Extracellular vesicles were extracted and their universal markers were used for identification.The results showed that extracellular vesicles can be used as a transportation route for EGFL6 to be transported to the outside of the cell.The results of Transwell experiment,chicken embryo allantoic membrane experiment and tubule formation experiment showed that FMNL2 regulates the migration and angiogenesis of HUVEC through EGFL6 paracrine,and the difference is statistically significant(P<0.05).The nude mouse subcutaneous tumor model and the immunohistochemical results of subcutaneous tumor showed that FMNL2 promoted angiogenesis in vivo through EGFL6,and the difference was statistically significant(P<0.05).The results of tail vein lung metastasis model and cecal orthotopic implant liver metastasis model showed that FMNL2 promoted colorectal cancer lung and liver metastasis through EGFL6,and the difference was statistically significant(P<0.05).Western blot results of paired colorectal cancer tissues showed that the expressions of FMNL2,EGFL6,and CD31 were positively correlated.The results of immunohistochemistry showed that the expression of FMNL2,EGFL6,and CD31 in colorectal cancer tissue was significantly higher than that in normal intestinal mucosal epithelial tissue,and they were correlated at the same location.4.EGFL6 can bind to the cytoskeletal membrane protein CKAP4 in HUVCEC,and FMNL2 can act on endothelial cells via EGFL6 paracrine to activate the ERK signaling pathway and promote the occurrence and development of colorectal cancer.Conclusion1.FMNL2 promotes the occurrence and development of colorectal cancer by stimulating tumor angiogenesis;2.There is a direct interaction between FMNL2 and EGFL6;3.FMNL2 can act on endothelial cells via EGFL6 paracrine to activate the ERK signaling pathway,promote angiogenesis,and promote the occurrence and development of colorectal cancer;... |
