| Backgroud and purposeColorectal cancer(CRC)is one of the most common gastrointestinal malignancies,its pathogenesis is concealed.The metastatic rate was high by the time it was first diagnosed.Metastasis is the major cause of CRC patients" death.Chemotherapy plays an important role in the treatment of metastatic CRC patients.L-OHP,5-FU and LV are commonly used chemotherapy regimens for metastatic CRC patients.However,treatment often failed due to CRC drug resistance.Therefore,it is of vital significance to explore the mechanism of metastasis and drug resistance of CRC.Tumor microenvironment was related to tumor invasion,metastasis and therapy response.CAFs,major mesenchymal cells of tumor microenvironment,regulated tumor progression,recurrence,metastasis,and therapy resistance by secreting exosomes,cytokines and remodeling extracellular matrix.However,the functions and mechanisms of CAFs exosomes in CRC metastasis and 5-FU/L-OHP resistance remain unclear.This study will explore the role and potential mechanisms of CAFs in the regulation of metastasis and drug resistance to provide new strategies for the clinic prevention and treatment of CRC.MethodsPrimary cell culture of CAFs and NFs isolated from fresh CRC tissues and corresponding normal mucosa using enzyme digestion.Exosomes were isolated from CAFs derived or NFs derived conditioned medium(CAFs-CM or NFs-CM)using ultra-high speed centrifuge,and were verified by transmission electron microscope,nanosight technique,and western blot.Exosomal miRNAs associated with CRC metastasis and drug resistance were screened using microRNA microarray,and its downstream targets were further predicted and verified using dual-luciferase activity system,qpcr,and western blot.Transwell boyden chamber,matrigel,sphere formation,colony formation,CCK-8,TUNEL and flowcytometry experiments were used to investigate cell migration,invasion,sternness,proliferation and apoptosis;orthotopic implantation in ileocecal junction was used to explore the effect of CAFs-derived exosomes(CAFs-exos)on CRC liver metastasis,subcutaneous injection of CRC cells was used to investigate the role of CAFs-exos in 5-FU/L-OHP drug resistanceResutlsImmunofluorescence and western blot results showed that α-SMA,FAP and FSP-1 proteins were positively expressed in CAFs,and negatively expressed in NFs.In vitro experiments showed that CAFs-CM promoted cell invasion and induced 5-FU/L-OHP drug resistance of CRC.Then we isolated exosomes from CAFs-CM and NFs-CM using ultracentrifugation and found that CAFs-exos promoted invasion,metastasis,maintained cell stemness and induced drug resistance of CRC.MicroRNA microarray was performed to analyze CAFs-exos and NFs-exos.Combining with microarray,qPCR and literature review,we selected miR-92a for further research.MiR-92a was highly expressed in CAFs-exos,and the level of miR-92a was significantly increased after uptake of CAFs-exos by CRC cells,and it is statistic significant(P<0.01).Bioinformatics,dual-luciferase activity.Qpcr and western blot experiments showed that FBXW7 and MO AP1 were downstream targets of miR-92a.Function experiments showed that CAFs-exosomal miR-92a promoted invasion,metastasis,maintained cell stemness and induced 5-FU/L-OHP drug resistance by suppressing FBXW7 and MOAP1.Conclusions1.CAFs-exos promoted cell metastasis,maintained cell stemness and induced 5-FU/L-OHP drug resistance of CRC cells;2.CAFs exosomal miR-92a promoted metastasis,maintained cell stemness and induced 5-FU/L-OHP drug resistance of CRC cells by directly inhibiting FBX W7 and MOAP1.Innovation1.Revealing a new mechanism:CAFs derived exosomal miR-92a promoted invasion and metastasis,maintained cell stemness and induced 5-FU/L-OHP drug resistance of CRC cells by directly inhibiting FBX W7 and MOAP1.2.Proposing a new target:CAFs derived exosomal miR-92a can be used as a new target for the prevention of metastasis and drug resistance of CRC... |
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