Effect Of Exosomes From Colorectal Cancer Cells On Macrophage Function And Its Mechanism

Background and aim:Colorectal cancer is one of the most common malignant tumors of the digestive system,and its morbidity and mortality occupy the forefront of malignant tumors.The latest data in 2018 show that the incidence of colorectal cancer ranks the third in all kinds of malignant tumors around the world,the the mortality ranks the second.In China,the incidence and mortality of colorectal cancer are increasing year by year,and colorectal cancer increasingly poses a huge threat to human health globally.The occurrence,development,invasion,and metastasis of colorectal cancer are extremely complicated processes.Although the pathogenesis of colorectal cancer has been well defined in recent years,the role of tumor microenvironment playing in the development of colorectal cancer remains to be further studied.Tumor-associated macrophages are important components in the tumor microenvironment and function as an essential role in immune and metabolic regulation.Existing researches show that the functional status of tumor-associated macrophages is highly plastic,changing according to environmental changes.At present studies,the classic model of tumor-associated macrophages is to divide them into M1 and M2 type macrophages.M1 macrophages play roles in killing tumor cells and inhibiting tumor progression,and M2 macrophages play roles in promoting tumor development.Studies have shown that colorectal cancer cells can "domesticate" macrophages in tumor tissues and promote the differentiation of macrophages towards M2 macrophages.However,the mechanisms and key targets remain to be clarified.DDX39 belongs to the DEAD Box family of RNA helicases.This family is involved in innate immune recognition,intracellular signaling pathway regulation,nuclear epigenetic regulation,and RNA transcription and splicing.We have found that the expression level of DDX39 in macrophages in tumor tissues was significantly lower in colon cancer mouse models than that in healthy mice as we previously described.Clinical specimens show that the expression of DDX39 in tumor tissue was significantly lower than those of normal intestinal mucosa,adjacent tissues and adenoma tissues,and the low expression of DDX39 in tumor tissues was significantly associated with poor outcomes.Immunofluorescence experiments show that DDX39 co-localized with F4/80,the common macrophage marker,but did not co-localize with CD163,the M2 macrophage marker,and the expression levels of DDX39 are negatively correlated with that of CD163.These results suggest that DDX39 may be related to the functional status of tumor-assciated macrophages.In tumor tissue,the functional status of macrophages is regulated by tumor cells.Previous studies have shown that cancer-derived exosomes play an important role in this regulation process.Exosomes are membrane vesicles with a diameter between 30 and 100 nm,which are actively secreted by cells.They contain proteins,lipids,sugar complexes,and micro RNAs(miRNAs).Exosomes often play important roles in regulating the functions of other cells.In pancreatic and breast cancer,miRNAs in cancer-derived exosomes can regulate the function of macrophages.Based on the previous research of our team,this study was aimed to detect the expression level of DDX39 in macrophages with different functional states and silencing with the expression of DDX39 in macrophages to explore the function roles of DDX39 on macrophages.On the other hand,our team aimed to explore the effects of colorectal cancer-derived exosomes on macrophage.In addition,this study combined with bioinformatics analysis to explore the signaling pathways and physiological processes that DDX39 may participate in,as well as the key miRNA regulating the differentiation of macrophages.Methods:This study consists of two parts.The first part mainly explored the effects of DDX39 on macrophage differentiation.We induced the human monocytic leukemia cell line THP-1 to differentiate into M1 and M2 type macrophages,and detected the expression level of DDX39 in mcrophages of two funcitonal states via RT-PCR technology,Western Blot technology and flow cytometry.To knock down the expression level of DDX39,we introduced the sh RNA plasmids which targets at the DDX39 locus into macrophage cells,and then detected the marker molecules of M1 and M2 macrophages by RT-PCR technology,ELISA technology and flow cytometry in order to explore the differentiation of macrophages.At the same time,CCK-8 was used to detect the proliferation ability of colorectal cancer cell line HT-29 after cultivated by conditioned medium from DDX39-konckdown macrophages.In this part,we also analyzed the differentially expressed genes between M1 and M2 macrophages in the GEO database,and performed enrichment analysis of GO and KEGG pathways to explore the signaling pathways that DDX39 may participate in.The second part mainly explored the effects of colorectal cancer-derived exosomes on macrophage differentiation and DDX39 expression.We first constructed a co-culture system of colorectal cancer cells and macrophages,and detected the marker molecules of M1 and M2 macrophages to explore the differentiation of macrophages via RT-PCR technology.We used ultracentrifugation to collect exosomes from the culture supernatant of HT-29,a colorectal cancer cell line,and used transmission electron microscopy,size distribution detection,and Western Blot technology to identify exosomes.After that,we carried out co-incubation experiments of exosomes and macrophages,and detected the phagocytosis of exosomes by macrophages using fluorescent staining.After determining that the exosomes were taken up by macrophages,we detect the marker molecules of macrophages by RT-PCR technology,ELISA technology and flow cytometry,thereby exploring the differentiation of macrophages.In addition,we also detected the expression of DDX39 in macrophages after exosomes treatment by RT-PCR technology and Western Blot technology.In this part,we analyzed the differential expression of miRNAs in the peripheral blood exosomes between colorectal cancer patients and healthy volunteers in the GEO database as well,and combined with Target Scan database to predict miRNAs that may be involved in the regulation of DDX39 expression,so that we may can discover the key miRNA in exosomes that may be involved in regulating macrophage differentiation.Results:In the first part,we illustrated the expression level of DDX39 in M1 macropages was higher than that of M2 macrophages in both m RNA and protein level,which were confirmed by RT-PCR and Western Blot.The RT-PCR and Western Blot results showed that sh DDX39 plasmid successfully knocked down the expression of DDX39 in macrophages;RT-PCR results showed that when the expression level of DDX39 decreased,the M1 macrophage markers,TNF-? and IL-1?,were decreased,the M2 macrophage marker while CD206,was increased.In line with that,when the expression of DDX39 decreased,the secretion of IL-12,a marker of M1 macrophages,decreased,while the secretion of IL-10,a marker of M2 macrophages,increased,which were demonstrated by ELISA.Flow cytometry results showed that when the expression level of DDX39 decreased,the proportion of CD206 + macrophages increased.The results of the CCK-8 experiment suggested the proliferation ability of HT-29 was strengthened after cultivated by conditioned medium from DDX39-konckdown macrophages.GEO data analysis results showed that there were 1586 differentially expressed genes between M1 and M2 macrophages.Gene enrichment analysis showed that genes up-regulated in M1 macrophages were mainly involved in the process of natural immunity,and genes that were up-regulated in M2 macrophages mainly participated in immune regulation.In the second part,RT-PCR results showed that after co-culture with HT-29 cells,the expression of DDX39 in macrophages was decreased at the m RNA level,as well as the expression of TNF-? and IL-1?,while the expression of CD206 increased.The disc-shaped morphology of the exosomes which were collected from the medium of HT-29 was observed by transmission electron microscopy.The particle size analysis showed that the size of the exosomes was mainly concentrated in the range of 40-100 nm.The results of Western Blot showed the expression of CD63 is highly expressed in exosomes.In a co-incubation experiment of exosomes and macrophages,fluorescent dye-labeled exosomes were co-cultured with macrophages for 24 hours,and fluorescence microscopy revealed that exosomes were engulfed by macrophages.RT-PCR results showed that after macrophages engulfed HT-29-derived exosomes,the expression levels of TNF-? and IL-1?,the M1 macrophage markers,decreased,while level of CD206,the M2 macrophage markers,increased,and the expression of DDX39 was down-regulated.The ELISA results showed that the secretion of IL-12,a marker of M1 macrophages,decreased,while the secretion level of IL-10,a marker of M2 macrophages,increased.Flow cytometry results showed that the proportion of CD206 + macrophages increased.The results of RT-PCR and Western Blot indicated that the expression of DDX39 was down-regulated after macrophages engulfed HT-29-derived exosomes.GEO data analysis results found that a total of 45 miRNAs were highly expressed in the exosomes from peripheral blood of colorectal cancer patients compared with healthy volunteers.And among them,hsa-miR-654-5p and hsa-miR-133 a have the potential to regulate DDX39 in macrophages.Conclusion:1.DDX39 has an impact on the differentiation direction of macrophages.When the expression level of DDX39 is reduced,macrophages differentiate into a direction that promotes tumor progression.2.Colorectal cancer-derived exosomes can promote the differentiation of macrophages into the direction of promoting tumor progression and reduce the expression level of DDX39.And hsa-miR-654-5p and hsa-miR-133 a may be key miRNA involved in the regulation of macrophage differentiation direction.