Mechanism Of Cancer-derived Exosomal Mir-25-3p In Regulating Pre-metastatic Niche Formation By Inducing Vascular Permeability And Angiogenesis

Background and purposeMetastasis is the main cause of death in clinical colorectal cancer patients.Primary tumor provides a favorable microenvironment in distant organs or tissues for subsequent metastasis,also known as pre-metastatic niche,which is formed by the interactions of primary tumor-derived exosomes and various cytokines,tumor-mobilized bonemarrow-derived cells(BMDCs),and the local stromal components.And exosome is widely regarded as an important tigger in the formation of pre-metastatic niche induced by primary tumors.Characteristics of the Pre-metastatic Niche:immunosuppression,inflammation,angiogenesis and vascular permeability,lymphangiogenesis,organotropism,reprogramming.We foucsed on the characteristics of angiogenesis and vascular permeability.In previous work,we found that miR-25-3p was correlative with metastasis via miRNA microarray,and the mRNA level of miR-25-3p in CRC parenchyma cells was positively related with that in the adjacent vascular endothelial cells.Therefore,we speculated that miR-25-3p could be transferred from CRC cells to adjacent vascular endothelial cells,or to vascular endothelial cells of the distant organs through the blood circulation,and regulate the angiogenesis and vascular permeability in Pre-metastatic Niche.This study aims to reveal a new mechanism of miR-25-3p in the formation of pre-metastatic niche and metastasis of CRC and provide a potential marker for prediction of CRC metastasis.MethodsIn this study,exosomes were purified from CRC-derived conditioned media or serum of CRC patients by ultra-high speed centrifuge,and were verified by transmission electron microscope and WB.Fluorescence tracer technique,exosomes PKH67 staining,exosomes uptake inhibitor AnnexinV and RT-qRCR experiments were used to investigate the uptake ability of exosomes.Transwell,in vitro permeability assay,CCK-8 cell proliferation assay and tube formation assay were carried out to detect vascular permeability and angiogenesis.miR-25-3p downstream targets were further predicted and verified by using dual-luciferase activity system.The expression of miR-25-3p downstream targets were detected by WB and IF experiments.After the injection were with 5?g of exosomes via tail vein,mice liver and lungs of nude mice were removed for frozen sections to observe the vascular permeability and angiogenesis.After the injection with 5?g of exosomes via tail vein,nude mice were injected with CRC cells via tail vein or spleen to observe the metastasis.ISH,IHC and RT-qPCR experiments were performed to detecte the expressions of miR-25-3p and its downstream targets in CRC patient tissues and serum.Results1.HUVEC could uptake CRC cell-derived exosomes,but CRC cells could not uptake HUVEC-derived exosomes.2.miR-25-3p could be transferred from cancer cells to HUVEC by exosomes.3.miR-25-3p promoted migration,proliferation,vascular permeability and angiogenesis of HUVEC in vitro.4.KLF2 and KLF4 were the downstream target genes of miR-25-3p.5.CRC cell-derived exosomal miR-25-3p down-regulated the expressions of KLF2,KLF4,zo-1,occludin and claudin5,up-regulated the expression of VEGFR2 in HUVEC and promoted proliferation,vascular permeability and angiogenesis of HUVEC.CRC cell-derived exosomal miR-25-3p promotes CRC metastasis by inducing vascular permeability and angiogenesis in pre-metastatic niche.6.The levels of miR-25-3p in circulating exosomes from CRC patients with metastasis was higher than those without metastasis and the healthy population.Moreover,the expression of miR-25-3p in circulating exosomes from postoperative CRC patients was significantly lower than that before surgery.The levels of miR-25-3p in circulating exosomes and CRC tissues were positively correlated.The level of miR-25-3p was negatively correlated with the expressions of KLF2 and KLF4 in CRC tissues.Conclusion1.This study unveils a novel mechanism of pre-metastatic niche formation that CRC cells-derived exosomal miR-25-3p could promote pre-metastatic niche formation and CRC metastasis.2.miR-25-3p derived from serum exosomes of CRC patients can be a potential biomolecular marker to predict the risk of CRC metastasis.