| Hypoxia microenvironment is an important part of tumor microenvironment,which has been proved to be closely related to the occurrence and development of malignant tumors.The tumor cell secreted exosomes(TDES)are considered to be the"carrier" of tumor metastasis.In the tumor microenvironment under the condition of hypoxia,the exosomes have certain significance for tumor.However,the specific regulatory role and potential mechanism of exosomes in anoxic microenvironment are still unclear.According to the research background and current situation,tumor cells can survive through epithelial mesenchymal transition(EMT)in hypoxia microenvironment.MicroRNAs,the contents of exosomes,affect cell proliferation,invasion and metastasis.The regulation of EMT in hypoxic tumor microenvironment is related to the content and stability of telomerase,but its mechanism is not clear.According to the bioinformatics and clinical validation,the binding miRNAs with human telomerase reverse transcriptase(hTERT)were screened in this study.The inhibition of EMT in vitro and in vivo and in vitro mediated by miR-1255b-5p was studied on the whole and cell level.We found that the hypoxia microenvironment directly affected the content of miR-1255b-5p.Under the hypoxia condition,the exocrine miR-1255b-5p secreted by colorectal cancer cells decreased,while the transmission of miR-1255b-5p between cells decreased.Interference with miR-1255b5p can promote EMT,colorectal tumor progression and liver metastasis.This study explored the specific molecular mechanism of cell proliferation,invasion and migration.This topic mainly includes the three parts,as the following:Part Ⅰ:Prediction and identification of exocrine miRNA in colorectal cancerBackground Colorectal cancer,the third most common cancer in the world,is one of the main causes of cancer death.Metastasis,as a multi-step and complex process,is the main cause of colorectal cancer related death.EMT occurs before metastasis,which is related to the poor prognosis of colorectal cancer treatment.Hypoxia microenvironment promotes the EMT process of many kinds of malignant tumors,which is the driving factor for tumor invasion and distant metastasis.Almost all cells will secrete exosomes after being stimulated(external pressure,changes of cell microenvironment,etc.)and distribute in different types of biological liquids,such as breast milk,urine,ascites and blood,etc.Because they are distributed in different types of biological fluids,and change with the change of the external micro environment.The miRNAs carried by tumor derived exosomes are closely related to the development and metastasis of tumor cells.In hypoxic microenvironment,exosomes secreted by tumor cells also play an important role in tumor invasion,proliferation,metastasis and telomerase.However,the relationship between exosomes and their specific mechanisms have not been fully elucidated.Methods The different miRNAs were screened by bioinformatics method,the exosomes were extracted from the serum samples of patients with colorectal cancer and normal people by the kit,and the exosomes were photographed by transmission electron microscopy,and the CD63,CD81 and TSG101 exosomes were detected by Western blot.MiRNAs were extracted from two kinds of exosomes.The expression of miRNAs in serum of colorectal cancer patients and normal people was detected by RT-PCR.Finally,we analyzed the relationship between the expression of exocrine miRNA and clinicopathological parameters in patients with colorectal cancer.Results Through preliminary bioinformatics screening and sequencing data(gse39833 data set),we identified three miRNAs(miR-1182,one binding site;miR-1255b-5p,three binding sites;miR-197-3p,two binding sites)of the 3’UTR binding site of hTERT.The expression level of mir-1255b-5p in colorectal cancer patients was significantly lower than that in healthy people,which was consistent with the relative expression of bioinformatics sequencing data.The morphology of exosomes was detected by transmission electron microscopy,and exosomes markers were detected by Western blotting.The feasibility of successfully separating exosomes from clinical serum samples was verified,which indicated that these exosomes were almost completely extracted from serum,but the remaining serum after extraction of exosomes did not exist.Considering the trend of miRNA expression and the number of binding sites with hTERT gene,we selected miR-1255b-5p as the target of follow-up study and preliminarily identified it as tumor suppressor miRNA.Clinical correlation analysis showed that the expression of miR-1255b-5p in exosomes of patients with colorectal cancer was significantly correlated with tumor stage and CA19-9 level.The later the tumor stage,the lower the expression level of miR-1255b-5p.MiR-1255b-5p was negatively correlated with the progression of colorectal cancer.Conclusions MiR-1255b-5p is a tumor suppressor gene,which can target hTERT.The expression level of miR-1255b-5p in exosomes secreted by patients with colorectal cancer is related to the clinical stage and CA19-9 level of patients,which can affect the progress of tumor and the prognosis of patients with colorectal cancer.Part Ⅱ:The mechanism of tumor cell EMT promoting proliferation,invasion and migration induced by exosomes from anoxic colorectal cancer cellsBackground Exosomes can play an important role in tumor invasion and metastasis by regulating EMT.The invasion and metastasis of tumor cells are further influenced by the "cross talk" between exosomes and surrounding cells.However,the interaction and mechanism of exosomes secreted by colorectal cancer cells during EMT under hypoxia are still unclear.Methods The exosomes isolated from the supernatant of colorectal cancer cells were isolated and identified by ultracentrifugation.A three-dimensional co culture model of exosomes and cells was established.The potential mechanism was further revealed by GST pulldown,double luciferase detection and RNA stabilization experiments.Results First,the exosomes secreted by colorectal cancer cells in normoxia/anoxia culture were isolated and identified,and a three-dimensional co culture model of exosomes and cells was established.The results showed that the exocrine miR-1255b5p secreted by colorectal cancer cells could induce EMT and promote the proliferation,invasion and migration of colorectal cancer cells.The mechanism study shows that miR-1255b-5p can promote the degradation of target gene by combining with 3’UTR of hTERT,which confirms the negative regulation relationship between miR-1255b-5p and hTERT;the exocrine miR-1255b-5p secreted by colorectal cancer cells in hypoxia microenvironment can regulate Wnt/β-catenin signal pathway to induce EMT of colorectal cancer cells,promote the proliferation and invasion of tumor cells And migration,while improving the length and activity of telomerase.Conclusion The relationship between miR-1255b-5p and hTERT is negative;the exocrine miR-1255b-5p secreted by colorectal cancer cells in anoxic microenvironment regulates Wnt/β-catenin signal pathway to induce EMT of colorectal cancer cells,promotes tumor cell proliferation,invasion and migration,and improves telomerase length and activity.Part Ⅲ:Effects of exosomes from anoxic colorectal cancer cells on subcutaneous tumorigenesis and liver metastasis in nude miceBackground In the previous experiment,we found that the exocrine miR-1255b-5p secreted by anoxic colorectal cancer cells could induce EMT to promote the proliferation,invasion and metastasis of colorectal cancer cells.Next,we used the methods of subcutaneous tumorigenesis and tail vein injection in vivo to verify the effect of exocrine miR-1255b-5p secreted by colorectal cancer cells in anoxic microenvironment on the subcutaneous tumorigenesis and liver metastasis of colorectal cancer cells.Methods The effect of exocrine miR-1255b-5p secreted by colorectal cancer cells in anoxic microenvironment on the subcutaneous tumorigenesis of colorectal cancer was evaluated by nude mice subcutaneous tumorigenesis model The effects of exosomes miR-1255b-5p secreted by colorectal cancer cells in anoxic microenvironment on the EMT status of colorectal cancer cells in nude mice were evaluated by blotting,H&E staining and immunohistochemical staining.The effects of exosomes miR-1255b-5p secreted by colorectal cancer cells in anoxic microenvironment on the liver metastasis of colorectal cancer cells were verified by tail vein injection.Results The exocrine miR-1255b-5p secreted by colorectal cancer cells in anoxic microenvironment can promote the growth of subcutaneous tumorigenesis of nude mice by regulating the EMT status of colorectal cancer cells,at the same time,it can enhance the proliferation of tumor cells and promote the liver metastasis of colorectal cancer cells.Conclusion The exocrine miR-1255b-5p secreted by colorectal cancer cells in anoxic microenvironment regulates Wnt/β-catenin signaling pathway to induce EMT of colorectal cancer cells and promote the growth of subcutaneous tumorigenesis in nude mice.It also promotes the proliferation of subcutaneous tumorigenesis and liver metastasis of colorectal cancer cells. |
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