| Introduction:Colorectal cancer(CRC)is the fourth most deadly cancer in the world.Surgical treatment is currently the main treatment for each CRC and the only option for radical treatment of CRC.However,most patients have a clear diagnosis and the tumor has invaded the large blood vessels or distant metastasis when the surgery is proposed,and the 5-year survival rate remains at about 10%for a long time.Therefore,clarifying the internal mechanism of the biological characteristics of high-invasion and metastasis of CRC is of great significance for improving the survival rate of CRC patients.Neutrophils mainly infiltrate into tumor epithelial cells.A large number of previous studies have shown that there are a large number of tumor-associated neutrophils(TANs)in cancerous tissues and the large number of neutrophil infiltration is related to the poor prognosis of cancer patients.The transforming growth factor-β(TGF-β)secreted by tumors tends to promote the phenotypic differentiation of TANs into N2,and the infiltration of N2 neutrophils is thought to be related to the metastasis and spread of tumors.However,little is known about the effect of N2 neutrophils on the malignant phenotype of CRC.Epithelial-mesenchymal transition(EMT)is a reversible cellular program that transforms polarized epithelial cells into mesenchymal cells and promotes cell migration.EMT is an important cellular mechanism in embryonic development,tissue repair,and disease by promoting the formation of gastrectomy,neural crest stratification,and the production of a variety of cells and tissue types.EMT is abnormally activated under pathological conditions such as organ fibrosis and cancer.In tumors,cancer cells use EMT to promote their separation from the primary tumor and spread into the blood,obtain the aggressive phenotype of tumor metastasis progression,and colonize metastases through mesenchymal epithelial transformation again.Exosomes are small lipid bilayer vesicles formed by the invagination of intracellular lysosomal particles.Because they can directly participate in the transmission of information and transport of substances between cells,they are considered to intercellular communication and immune regulation.An important carrier of circulating biological markers for disease diagnosis and prognosis.There is increasing evidence that exosomes released by tumor-associated immune cells can promote tumor growth and metastasis.However,there is still lack of research on the mechanism of N2 neutrophil-derived exosomes affecting the phenotype of malignant tumors.micro ribonucleic acid(miRNA)is a kind of endogenous small molecular single-stranded RNA without coding function,with only 20-25 nucleotides They were first discovered in Caenorhabditis elegans and can be found in the ribonucleic acid-induced silencing complex.The stability and/or translation of a target gene is regulated by base pairing between its 5’-UTR seed sequence and the 3’-UTR recognition element of the target mRNA.Studies have shown that miRNA play a key role in physiological and pathological processes,including cell differentiation,development,metastasis,and tumor formation.Studies have confirmed that there is abnormal expression of miRNA in the process of tumor occurrence and development.miRNA is involved in the regulation of tumor process by promoting or inhibiting the EMT process of cancer cells.However,with the deepening of research on tumor microenvironment(TME),whether tumor-related immune cells,especially tumor-related neutrophils,can affect the malignant biological behavior of tumors through exosomes and the specific molecular mechanisms have become a new research direction.Objectives:By comparing the degree of neutrophils infiltration in CRC tissues of patients with CRC,this study explored the internal relationship between neutrophils infiltration and CRC metastasis.To investigate the effect of miR-4780 in N2 neutrophils exosomes on the malignant biological behavior of CRC,and to verify its targeting relationship with SOX11 and its regulatory effect.Methods:In this study,30 CRC patients aged 36-82 who were hospitalized in the First Affiliated Hospital of Kunming Medical University from January 2017 to October 2019 were selected as the research objects.All participants obtained informed consent and signed.The Cancer Imaging Archive(TCIA)database was used to analyze the infiltration of different immune cells in CRC TME,and the internal relationship between neutrophils infiltration and CRC metastasis was explored by comparing the infiltration degree of neutrophils in clinical specimens.N2 neutrophils were obtained from human myeloid leukemia cell line NB-4 cells induced by all-trans retinoic acid and TGF-β successively.Exosomes from neutrophils culture medium supernatant were extracted by low temperature and ultra-high-speed centrifugation combined with density gradient centrifugation.The size and morphology of exosomes were observed by Hitachis-3000N cryo-scanning electron microscopy,and the protein components of exosomes were identified by molecular biological methods.NO or N2 neutrophil-derived exosomes were co-cultured with COLO205 cells,and the effects of NO or N2 neutrophil-derived exosomes on CRC cells were investigated by Transwell,cell clone formation assay,CCK-8,Western blot,and RT-qPCR.Differential miRNAs of exosomal origin in N2 neutrophils were screened by exosomal miRNA microarray assay,and their effects on malignant phenotypes of CRC cells were verified in vitro by exogenous regulation of differential mRNA expression.The targeting and regulation of miR-4780 in N2 neutrophil exosomes to EMT-related protein SOX 11 were investigated by dual luciferase assay.At the same time,the model of CRC metastasis in nude mice was established,and the liver metastasis of CRC in mice was evaluated by in vivo imaging technology.The EMT-related proteins were detected by Western blot and immunofluorescence,and the regulation of the EMT process of CRC by N2 neutrophil-derived exosome miR-4780 was confirmed.Results:1.Among the 30 CRC patients aged 34-81 years,there were 19 males and 11 females,with an average age of 61.32±11.40 years.2.Neutrophils were relatively enriched in CRC,hepatocellular carcinoma,gastric adenocarcinoma,bladder carcinoma and lung adenocarcinoma.In addition,the infiltration ratio diagram of CRC TME immune cells in the database showed that neutrophils accounted for 32%,and were the most infiltrated immune cells in CRC.It suggested that neutrophils were enriched in CRC tissue.3.The expressions of CD11b and CD66b in metastatic CRC tissues were significantly higher than those in non-metastatic CRC tissues,suggesting that the enrichment of neutrophils in CRC tissues may be related to CRC metastasis.4.Compared with NO-ExO co-culture,COLO205 co-culture with N2-ExO significantly enhances proliferation,migration,and invasion,and decreases apoptosis rate.5.The differential miRNAs of exosome origin were screened by exosome miRNA microarray detection,and 18 miRNAs were matched with P<0.05 was the requirement for a statistical significance difference.6.Overexpression of N2 neutrophil-derived exosome-derived miR-4780 promotes the invasive behavior of COLO205 cells and the occurrence of EMT,promoting cancer metastasis.7.SOX11 is a regulatory target of miR-4780,and N2 neutrophil-derived miR-4780 can be uptake by COLO205 cells and regulate intracellular SOX 11 expression.8.N2 neutrophil-derived exosomes can inhibit the expression of SOX11 in CRC cells through miR-4780,and promote liver metastasis of CRC and aggravate liver injury.Conclusion:In CRC,miR-4780 from N2 neutrophil-derived exosomes can promote EMT and malignant phenotype of CRC by targeting down-regulation of SOX11,and this regulatory pathway may have potential value in the diagnosis and treatment of CRC. |
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