| Objective:Colorectal cancer is a worldwide tumor and the fastest growing digestive tract malignancy in China.At present,the main treatment for colorectal cancer is still surgical resection.The prognosis of early colorectal cancer is relatively good,but most patients are advanced at the time of treatment,and most patients have metastasized.Metastatic colorectal cancer is one of the most common causes of cancer-related death,and research on its mechanism of development has received extensive attention from researchers.The metastasis of colorectal cancer is affected by many factors,among which angiogenesis and self-metastasis play an important role in the rapid growth and metastasis of colorectal cancer.Early tumors are less prone to angiogenesis because of their slow production and the consumption of very little oxygen and nutrients.If there is no angiogenesis in the early stage of the tumor,the tumor growth will not exceed 3mm~3.In the late stage of the tumor,as the tumor cells continue to grow,the tumors continue to increase,resulting in the lack of oxygen and nutrients inside the tumor.This hypoxic and nutrient-deficient extracellular environment can stimulate tumor cells to secrete a variety of angiogenesis related factors and other Substance,induces the formation of new blood vessels and the metastasis of the tumor itself,providing sufficient oxygen and nutrients for the rapid production of cells inside the tumor.In metastatic colorectal cancer,under the hypoxic environment,the mechanism of neovascularization and metastasis of colorectal cancer cells is crucial for the development of anti-tumor targeted drugs.Exosomes are lipid bilayer vesicles of about 30nm-100nm in size that originate from intracellular polycytes and are actively secreted by cells.Almost all cells can produce exosomes.In the process of its occurrence and development,tumor cells continuously release tumor-specific exosomes to the extracellular space,and can regulate the tumor microenvironment through the biological substances such as DNA,RNA and protein of the tumor cells they carry.The study found that tumor cell-derived exosomes can stimulate the regeneration and self-transmission of blood vessels,mainly through the uptake of tumor cell-derived exosomes by endothelial cells and normal cells,especially in tumor tissue hypoxia-stimulated exosomes.Increased circumstances.Evidence suggests that exosome derived from colorectal cancer cells plays an important role in the biological behaviors such as invasion,metastasis,angiogenesis and immune regulation of colorectal cancer through small RNA,messenger RNA or protein.However,the function and mechanism of cancer from exocytic cells derived from hypoxic colorectal cancer cells have not been reported so far.A variety of signaling pathways are involved in tumor angiogenesis and metastasis,and the role of Wnt/?-catenin pathway changes in various cancers has been reported.The activated Wnt protein further recruits Axin protein,which transforms the latter from an inhibitory complex to a stimulatory signalsome,further promoting downstream proteins.A variety of proteins in the Wnt family are involved in the development of tumors.However,the molecular mechanisms by which Wnt family proteins promote angiogenesis and metastasis in colorectal cancer still require further exploration.This study intends to further explore the mechanism of exocytosis derived from hypoxic colorectal cancer cells to promote angiogenesis and metastasis of colorectal cancer.Through co-culture with vascular endothelial cells,co-culture of normoxic cells,etc.,through CCK8,transwell experiments,Trace experiments and other means to confirm the role of exocytosis derived from hypoxic colorectal cancer in angiogenesis and metastasis of colorectal cancer.On this basis,in-depth study of whether the Wnt/?-catenin pathway plays a role in this,through in vitro cell experiments and in vivo animal experiments,to determine the role of exocyx derived from hypoxic colorectal cancer cells on tumor angiogenesis and metastasis.To lay the foundation for further development of anti-tumor drugs.Methods:In this study,HT29 and HCT116 two colorectal cancer cell lines were selected for normal oxygen and hypoxia culture.After extracting exosomes,they were co-cultured with vascular endothelial cells.CCK8 was used to detect the proliferation of vascular endothelial cells.Transwell assay was used to detect migration.After the ability changes,and then inhibited the expression of exosomes,the exosomes-vascular endothelial cell system was also constructed to observe whether the proliferation and migration ability changed with the decrease of exosomes.The activation of Wnt-?-catenin is regulated intracellularly,confirming the role of this pathway in angiogenesis promoted by hypoxic colorectal cancer exosomes.Screening pro-angiogenic factors in exocrine-derived exocrine cells,and knocking down this factor to determine whether this factor mediates the proliferation and migration of vascular endothelial cells,and whether the increase of this factor is related to hypoxia..At the same time,the co-culture of hypoxic colorectal cancer-derived exosomes and normoxic colorectal cancer cells was carried out to verify the promotion of the above-mentioned key molecules in the invasion and metastasis of normoxic colorectal cancer cells,and the Wnt-?-catenin pathway was identified in the process.Results:1.Exosomes derived from hypoxic colorectal cancer cells induce increased proliferation and migration of endothelial cells.2.Inhibition of exocrine secretion of colorectal cancer cells inhibits proliferation and migration of endothelial cells.3.Exosomes derived from hypoxic colorectal cancer increase the proliferation and migration of endothelial cells through Wnt4.4.Hypoxia stimulates Wnt4 up-regulation in exosomes in a HIF-1?-related manner.5.Proliferation and migration of endothelial cells requires Wnt4 activation of the?-catenin signaling pathway by exosomes.6.Exosomes derived from hypoxic colorectal cancer cells increase the migration and invasion of normal colorectal cancer cells.7.Inhibition of colorectal cancer exocytosis reduces the migration and invasion of hypoxic colorectal cancer cells induced by exosomes derived from hypoxic colorectal cancer cells.8.The increase in migration and invasion of exoccal colon cancer cells induced by exocrine-derived exocrine cancer cells is dependent on HIF1?.9.Exosomes Wnt4 promote migration and invasion induced by exosomes derived from hypoxic colorectal cancer cells.10.Exosomal Wnt4-activated?-catenin is required for the migration and invasion of normoxal colorectal cancer cells.11.In vitro experiments confirmed that exogenous Wnt4 derived from hypoxic colorectal cancer cells promoted the growth and angiogenesis of colorectal cancer.Conclusion:1.Exosomes released by hypoxic colorectal cancer cells can promote the proliferation and migration of vascular endothelial cells.In addition,the body is rich in Wnt4.Exosomes of Wnt4 increase nuclear translocation of?-catenin in endothelial cells.Activation of the?-catenin signaling pathway is critical for the proliferation and migration of vascular endothelial cells.In vivo animal studies have further revealed that exosomes that affect the origin of hypoxic colorectal cancer cells have a role in promoting tumor growth and angiogenesis.That is,under hypoxic conditions,colorectal cancer cells promote endothelial angiogenesis via exosomes-mediated Wnt/?-catenin signaling pathway,which may be a new target for the treatment of colorectal cancer.2.The exosomes released by hypoxic colorectal cancer cells enhance the migration and invasion of normal colorectal cancer cells.Exosomes derived from hypoxic colorectal cancer enhance the migration and invasion of normal colorectal cancer cells through Wnt4.Exosomes Wnt4 enhance the nuclear translocation of?-catenin in normoxal colorectal cancer cells.Activation of the Wnt-?-catenin signaling pathway is critical for the migration and invasion of normal colorectal cancer cells.That is,hypoxia can stimulate tumor cells to release Wnt4-rich exosomes,which are then endocytosed by oxygenated cells to promote metastasis.This study may provide new targets for the treatment of colorectal cancer,especially metastatic colorectal cancer. |
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