The Mechanism Of Exosomal C-KIT Derived From Mast Cells In Promoting The Growth And Metastasis Of Colorectal Cancer

Objective Colorectal cancer(CRC)is one of the most common malignant tumors of the digestive system cancer.The latest cancer statistics show that CRC accounts for about 10% of cancer incidence and mortality,and it is increasing year by year.The poor prognosis of CRC is mainly due to distant metastasis,and liver metastasis is the the main cause of death in patients.Therefore,the studies on the growth and metastasis of CRC is urgent.As one of the earliest tumor infiltrating cells,mast cells contain a large number of vesicle structures.Studies have shown that the vesicle belong to multivesicular bodies,and the bodies will release exosomes when they fuse with the cell membrane.It is reported that exosomes play an important role in the occurrence,development,invasion and metastasis of tumors.C-KIT,as a marker protein of mast cells,participates in intracellular signal transduction and regulates cell proliferation,growth and metastasis.We found that C-KIT positive mast cells were abundant in CRC tissues.and the C-KIT protein was detected in exosomes secreted by mast cells.Therefore,this study intends to explore exosomes secreted by mast cells are involved in regulating the growth and metastasis of CRC,and the effect of exosomal C-KIT on the proliferation,migration and invasion of CRC cells.Methods In this study,36 clinical specimens of CRC and adjacent normal tissues were collected,and the expression of mast cells and C-KIT in CRC and normal adjacent tissues was detected by toluidine blue staining,immunohistochemistry and Western blot.The correlation between C-KIT and clinical pathological data was analyzed.In vitro,mast cell exosomes were extracted by ultracentrifugation,electron microscopy,particle size analysis and Western blot were used to detect exosomes morphology,size and marker protein,and immunofluorescence were used to detect C-KIT and exosomal marker proteins.Uptake test was used to detect the exosomes were taken up by CRC cells.The sh RNA was used to downregulate C-KIT expression in mast cells.CRC cells were divided into control group(NC),exosomes group(Exosomes),and exosomes C-KIT sh RNA group(Exosomes C-KIT sh RNA).CCK-8 test,Edu test,cell scratches test and Transwell test were used to detect the effects of exosomal C-KIT secreted by mast cells on the proliferation,migration and invasion of CRC cells.Western blot was used to detect the expression of E-cadherin,N-cadherin and vimentin,and also verify exosomal C-KIT activates MEK/ERK signaling pathway to promote CRC cell growth.In vivo.the effect of exosomal C-KIT secreted by mast cells on the growth of transplanted tumors by injecting exosomes.Results By toluidine blue,immunohistochemistry and Western blot confirmed that the expression of mast cells and C-KIT was significantly higher in CRC tissues than in adjacent tissues.And C-KIT expression was positively correlated with CRC size and lymph node metastasis(P<0.05).By transmission electron microscopy confirmed that the exosomes have a clean background and like "flattened spherical structure".The size of exosomes was mainly distributed at 30-150 nm.Western blot detected the exosomal marker protein CD63,CD81,TSG101,Alix and C-KIT protein expression.Meanwhile,the C-KIT protein can co-localize with exosomal marker proteins CD63 and TSG101,and exosomes can be taken up by CRC cells.Compared with the control group and the exosomes C-KIT sh RNA group,the exosomes group can significantly promote the proliferation,migration and invasion of CRC cells(P<0.05).Western blot showed that the epithelial-mesenchymal transition(EMT)molecular markers N-cadherin and Vimentin protein were significantly increased after co-culture with exosomes,while Ecadherin protein were significantly decreased.At the same time,the expression of MEK and ERK phosphorylation were increased.In vivo.Exosomal C-KIT secreted by mast cells can promote the growth of CRC.Conclusion Our study results show that the exosomal C-KIT secreted by mast cells can be taken up by CRC cells,and promote the proliferation,migration and invasion of CRC cells.Besides,we further demonstrate that exosomal C-KIT can activate the MEK / ERK signaling pathway to promote the occurrence of EMT in CRC cells.Therefore,exosomal C-KIT secreted by mast cells may serve as a new target for CRC treatment.