Mechanism And Clinical Significance Of The Effect Of Exosomes Secreted By Colorectal Cancer On Tumor Invasion And Metastasis

Inflammatory cytokines are closely related to the occurrence and development of malignant tumors.Exosomes are considered to be "mediators" of solid tumor metastasis,and they play an important role in the development of many malignant tumors.However,the role of exosomes in inflammatory factor-mediated colorectal cancer metastasis and the specific regulatory mechanisms remain unclear.Based on the above research background and current situation,this study firstly used IL-6 to induce the epithelial mesenchymal transition(EMT)model in colorectal tumor cell lines,and extracted exosome miRNA from the supernatant before and after transformation for high-throughput sequencing,and obtained the differential gene mir128-3p.Furthermore,we conducted a series of cell biology,molecular biology and in vivo animal experiments to investigate the regulation of exosomal miR-128-3p on the EMT process of colorectal tumor cells and its specific molecular mechanism.Finally,we evaluated the correlation between the expression level of miR-128-3p in serum exosomes of patients with colorectal cancer and the clinical progress of patients with clinical serum samples and their clinical application value.This topic mainly includes the four parts,as the following:Part 1:Establishment of IL-6-induced EMT model of colorectal tumor cells and determination of differental miR-128-3p in exosomeObjective As the third most common malignant tumor in the world,colorectal cancer is one of the leading causes of tumor-related death.The leading cause of colorectal cancer-related death is metastasis.One of the mechanisms of colorectal tumor metastasis mainly involves cells before invasion.These cells show changes in epithelial structure and function,leading to further enhancement of motility and invasiveness.This process is called epithelial-mesenchymal transformation transition(EMT),which is associated with a poor prognosis for colorectal cancer treatment.Inflammatory factors are key factors that promote tumor growth and induce EMT in a variety of tumor cells.Exosomes are vesicles about 30-150nm in diameter secreted by cells.Almost all cells secrete exosomes after being stimulated(external pressure,changes in cell microenvironment,etc.),and they are distributed in different types of biological fluids,such as breast milk,urine,ascites and blood.MiRNAs carried by tumor-derived exosomes are closely related to the development and metastasis of tumor cells.In colorectal cancer,the microenvironmental changes of colorectal cancer cells caused by inflammatory factors,and the molecular mechanism by which exosomes miRNAs regulate tumor cell EMT in tumor microenvironment have not yet been elucidated.Methods The EMT model was constructed using IL-6 to induce colorectal cancer HCT116 cell line.Subsequently,Western Blot was used to identify EMT marker proteins.Exosomes were extracted from IL-6-induced and IL-6-uninduced tumor cell supernatant by ultrafast centrifugation method.Exosome morphology was photographed by transmission electron microscopy and exosome markers such as CD63,CD81 and TSG101 were detected by Western Blot to ensure successful exosome extraction.MiRNAs were extracted from the exosomes derived from the two cell supernatants,and differential miRNAs were found after high-throughput sequencing.Real-time fluorescence quantitative PCR(qRT-PCR)method was used for verification,and the results were consistent with the sequencing results.Bioinformatics was used to analyze the function of differential miRNAs,and miRNAs with high close correlation with EMT were selected for further research.Results After IL-6 induced colorectal cancer HCT116,E-cadherin expression was down-regulated,and Vimentin,N-cadherin and ZO-1 expression were up-regulated,indicating that the EMT model was successfully constructed.Under transmission electron microscopy,exosomes secreted by supernatant in the IL-6-induced group and the IL-6-uninduced group were vesicles with a diameter of about 30-150nm,and the expressions of CD63,CD81 and TSG101 were significantly higher than those of corresponding cell proteins,indicating the successful exosome extraction.High-throughput sequencing results showed that a total of 773 differential miRNAs were detected,of which 190 had statistical differences(pvalue<0.05).Among 190 differential miRNAs,16 miRNA highly correlated with exosomes were successfully screened by bioinformatics for real-time quantitative PCR verification,and one miR-128-3p highly correlated with exosomes and EMT was finally identified.Conclusions There are differences in miRNA expression in exosomes secreted by colorectal tumor cells with and without EMT,and miR-128-3p may play an important role in the development of colorectal tumor cells with EMT.Part Ⅱ:MiR-128-3p promotes proliferation,migration and invasion of colorectal tumor cells and its specific mechanismObjective Previous studies have shown that miRNAs play an important role in tumor invasion and metastasis by regulating tumor cell EMT.However,the role of miR-1283p in the EMT process of colorectal tumor cells and the specific molecular mechanism are still unclear.Methods A series of cell biology experiments were used in vitro to evaluate the effects of miR-128-3p on the EMT status,proliferation,migration and invasion ability of colorectal tumor cells.Further molecular biology experiments such as dual luciferase reporting experiments and RNA stability assays have revealed potential mechanisms.Results In vitro experiments show that overexpression of miR-128-3p can promote the proliferation,migration,invasion and EMT of colorectal tumor cells;in contrast,knockdown of miR-128-3p can inhibit the proliferation,migration,invasion and EMT of colorectal tumor cells.Further mechanism studies have confirmed that miR-128-3p regulates JAK/STAT3 and TGF-β/SMAD signaling pathways through its target gene FOXO4 to induce EMT in colorectal tumor cells and promote tumor cell invasion and metastasis.Conclusion MiR-128-3p induces EMT of colorectal tumor cells through its target gene FOXO4 to promote proliferation,migration and invasion;JAK/STAT3 and TGFβ/SMAD signaling pathways play a very important role in it.Part Ⅲ:Effect of exosomal miR-128-3p on EMT cascade in colorectal tumor cells in vitro and in vivoObjective Studies have confirmed that in the tumor microenvironment,tumor cells have "cross-talk" with surrounding cells through exosomes to further cause the invasion and metastasis of tumor cells.Therefore,it has not been clarified whether tumor cells with EMT can secrete exosomes to the surrounding cells to make them change so as to promote tumor invasion and metastasis.Methods In vitro experiments using the EMT of colorectal tumor cells secrete body and EMT in tumor cell co-culture model and a series of cell biology experiments to explore the occurrence of EMT in colorectal tumor cells secrete secrete body outside miR-128-3p on the surrounding has not yet occurred EMT EMT status,tumor cell proliferation,migration and the influence of the invasive ability.In vivo experiments,the effect of exosomal source miR-128-3p on subcutaneous tumorigenesis ability of colorectal tumors was evaluated by subcutaneous tumorigenesis model of nude mice and tail vein injection of exosomes,and the effect of exosomal source miR-128-3p on EMT status of colorectal tumor cells was further evaluated by qRT-PCR and Western Blot.Results In vitro experiments,after co-culturing exosomes secreted by miR-128-3p overexpressed colorectal tumor cells with ordinary colorectal tumor cells,compared with the control group,the proliferation,migration and invasion ability of receptor cells in the overexpressed group increased significantly,and EMT was promoted.After coculturing the exosomes secreted by miR-128-3p knocked down colorectal tumor cells with normal colorectal tumor cells,compared with the control group,the proliferation,migration and invasion ability of recipient cells in the knocked down group was significantly reduced,and EMT was inhibited.In vivo experiments,exosomes secreted by colorectal tumor cells that overexpressed miR-128-3p were injected into the tail vein of nude mice.After injecting the exosomes secreted by colorectal tumor cells that knock down miR-128-3p into the tail vein of nude mice,the growth rate and size of subcutaneous tumor formation were smaller than that of the control group.The results of qRTPCR and Western Blot showed that after injecting exosomes secreted by colorectal tumor cells overexpressing miR-128-3p into the tail vein of nude mice,tumor cells EMT in subcutaneous transplanted tumor tissues was promoted.After injecting the exosomes secreted by colorectal tumor cells that knocked down miR-128-3p into the tail vein of nude mice,the tumor cells EMT in the subcutaneous transplanted tumor tissues was inhibited.Conclusion Colorectal tumor cells with EMT can secrete exosome miR-128-3p and transmit it to surrounding tumor cells,so that it can also produce EMT and enhance the proliferation,migration and invasion ability of tumor cells,thus realizing the cascade amplification effect of EMT.Part Ⅳ:Correlation between serum exosomal miR-128-3p and clinical tumor progression in patients with colorectal cancerObjective In order to explore the correlation between exosomal miR-128-3p and the clinical progress of colorectal cancer patients,the clinical significance of exosome miR128-3p in colorectal cancer was revealed.Methods Firstly,serum samples from 66 patients with colorectal cancer were isolated by cryogenic centrifugation,and then serum exosomes were extracted by exosomes extraction kit.Then miR-128-3p expression level in exosomes were detected by realtime fluorescence quantitative PCR(qRT-PCR).Finally,the correlation between serum exosomes miR-128-3p,clinical pathological characteristics of patients,and clinical progress of patients was analyzed.Results Univariate analysis of factors such as gender,age,tumor type,tumor size,clinical stage,tumor grade,nerve invasion,vascular invasion,CEA,CA199 and other factors showed that serum exosome miR-128-3p,nerve invasion,vascular invasion and CA199 were independent factors affecting patients’ clinical stage.The expression level of miR-128-3p in serum exosomes was positively correlated with the clinical stage of colorectal cancer patients:the higher the expression level of miR-128-3p in serum exosomes,the later the clinical stage of the patients.Conversely,the lower the expression level of serum exosome miR-128-3p,the earlier the clinical staging of the patient.For the effective variables in single factor:nerve invasion,vascular invasion,clinical stage,CA199 multivariate binary Logistic regression analysis showed that clinical stage and vascular invasion of exosomal miR-128-3p expression level had important influencing factors.The later the clinical stage and the higher the nerve invasion degree were,the higher the exosome miR-128-3p expression level was,showing a positive correlation.Therefore,we believe that clinical staging and nerve invasion are important factors affecting the expression level of miR-128-3p in exosomes.The vascular invasion and CA199 expression level of exosomal miR-128-3p had a certain effect,but the effect was not obvious.Conclusion The expression level of miR-128-3p in serum exosomes was positively correlated with the clinical progress of colorectal cancer patients.Serum exosome miR128-3p has potential clinical application value in the diagnosis and treatment of colorectal cancer.