Exosomal MiR-210-3p Promotes Invasion Of Colorectal Cancer Cells And Bioinformatics Analysis Of Its Target Genes

Objective:Exosomes participate in biological processes such as tumor cells invasion,metastasis,and angiogenesis.Invasion and metastasis are the main cause of death in colorectal cancer(CRC)patients.It is of great importance to investigate the mechanism of CRC metastasis.Tumor derived exosomes are able to carry miRNAs into the receptor cells and are reported to be involved in tumor invasion,metastasis and angiogenesis.A number of researches have shown that miR-210-3p is up-regulated in lung cancer and renal cancer,and it can also regulate the proliferation,migration and invasion of breast cancer cells.However,whether the exosomes derived from CRC cells express miR-210-3p and its role in the invasion of colorectal cancer has not been reported.Therefore,this study aims to explore the role of exosomal miR-210-3p in the invasion of colorectal cancer cells and to analyze the predicted target genes by bioinformatic methods.Methods:1.Effect of miR-210-3p on the migration and invasion of SW480 cells:(1)The expression of mi R-210-3p in CRC was analyzed by dbDEMC and OncomiR database;(2)Quantitative real-time PCR(qRT-PCR)was used to detect the expression of miR-210-3p in CRC cell lines;(3)qRT-PCR was used to detect the expression of miR-210-3p after transfection of miR-210-3p mimics and inhibitor in SW480cells;Transwell migration and invasion assays were used to analyze the migration and invasion of SW480 cells after transfection of miR-210-3p mimics and inhibitor.2.Extraction and characterization of exosomes:(1)The exosomes derived from SW620 cells were extracted by ultracentrifugation;(2)The exosomes were morphologically characterized by transmission electron microscopy;(3)The expression of biomarkers of exosomes were analyzed by Western blot.3.Effect of exosomal miR-210-3p on recipient cells:(1)The expression of exosomal miR-210-3p derived from CRC cells was detected by qRT-PCR;(2)After incubating with exosomes derived from SW620 cells,the expression of mi R-210-3p in SW480 cells was detected by qRT-PCR;(3)The effect of exosmal miR-210-3p derived fromSW620 cells on the migration and invasion of SW480 cells was analyzed by Transwell assays.4.Prediction and bioinformatics analysis of target genes of miR-210-3p:(1)miRDB,TargetScan7.2,starBase and miRPathDB databases were used to predict target genes of miR-210-3p;(2)GO analysis and KEGG enrichment analysis were performed by DAVID;(3)String database was used to predict interactions between proteins encoded by the key target genes;(4)The expression of key target genes were analyzed by GEPIA.Results:1.Mi R-210-3p promotes migration and invasion of SW480 cells :(1)dbDEMC and OncomiR database showed that miR-210-3p was highly expressed in CRC,which was related to tumor metastasis and prognosis;(2)The expression of miR-210-3p in highly metastatic LoVo,HCT-116 cells was significantly higher than that in low metastatic SW480,CL187,HCT-8 cells;(3)SW480 cells migration and invasion significantly increased after transfection of miR-210-3p mimics,while decreased after transfection of miR-210-3p inhibitor,suggesting that mi R-210-3p can promote SW480 cell migration and invasion.2.Extraction and characterization of exosomes:(1)Exosomes derived from highly metastatic SW620 cells showed a typical goblet structure with diameter of about 60nm;(2)The specific exosomal markers CD63,CD9,and CD81 were highly expressed in the exosomes derived from SW620 cells.3.Exosomal miR-210-3p promote SW480 cell migration and invasion:(1)The expression of exosomal miR-210-3p secreted by highly metastatic CRC SW620 cells was significantly higher than that in low metastasis SW480 cells;(2)After incubated with exosomes derived from SW620 cells,the expression of mi R-210-3p in SW480 cells was significantly increased;and after incubated with exosomes derived from SW620 cells transfected inhibitor,the expression of miR-210-3p in SW480 cells was significantly reduced;(3)Transwell assays showed that migration and invasion of SW480 were elevated by incubation with exosomal miR-210-3p from SW620 cells.4.Prediction and bioinformatics analysis of miR-210-3p target genes:(1)A total of3035 target genes were predicted by miRDB,TargetScan7.2,starBase andmiRPathDB databases;(2)The functions of miR-210-3p target geneswere mainly enriched in biological processes such as cell migration,adhesion,protein metabolism.The enriched pathways include cancer pathway,HIF signaling pathway and Rap1 signaling pathway;(3)Interaction of proteins encoded by 25 target genes played a crucial role as analyzed by String database;(4)RGMA is significantly down regulated in clinical CRC tissues,and may be the target gene of miR-210-3p.Conclusion:1.miR-210-3p can promote CRC cells migration and invasion 2.miR-210-3p is highly expressed in exosomes derived from highly metastatic CRC cells,and enter the receptor cells through horizontal transmission,which can promote the migration and invasion.