Study On The Mechanism Of Exosome-Shuttling Micrornas In Human Esophageal Cancer Microenvironment

Background and Objects:China is one of the countries with both the highest incidence and mortality rates of esophageal cancer,which threatening the safety and quality of people's life.It has a great significance for the screening of esophageal cancer high-risk groups that are searching for biomarkers with high sensitivity and specificity for the early diagnosis of esophageal cancer.In this study,we selected the differentially expressed miR-21-5p and miR-93-5p in plasma samples from esophageal cancer patients and healthy volunteers as our research objects.Aim to(1)explore the biological effects of esophageal cancer cell-derived exosome-shuttling miR-21-5p and miR-93-5p to the recipient cells including esophageal cancer cells,human umbilical vein endothelial cells(HUVEC)and tumor-associated macrophages(TAM)in esophageal cancer microenvironment;(2)recognize the target genes and downstream signaling pathways of exosome-shuttling miR-21-5p and miR-93-5p;(3)preliminarily explore the related mechanisms of exosome-shuttling miR-21-5p and miR-93-5p and evaluate their diagnostic value for esophageal cancer.Methods:1.We isolated precipitates from the culture supernatant of esophageal cancer cells EC9706 following a classical gradient centrifugation and then ultracentrifugation protocol.The exosomes were identified with the precipitates using transmission electron microscopy and Western blot.The next-generation RNA sequencing technology was carried out to construct two small-RNA cDNA libraries from EC9706 cells and EC9706 cells-derived exosomes.2.Eighty-seven primary esophageal cancer patients and matching healthy volunteers were recruited with informed consent.Three pairs of plasma samples from them were random selected and prepared for microarray analysis.Combined with the Solexa high-throughput sequencing results,we used quantitative RT-PCR(qRT-PCR)for the validation of differentially expressed miRNAs in larger population samples.Bioinformatics analysis was adopted for the target gene prediction,then gene ontology and pathway analysis were performed The association between differentially expressed miRNAs and the risk of esophageal cancer were assessed by conditional logistic regression analysis.Meanwhile,the area under receiver operating characteristic curve was applied for the evaluation of their diagnostic value for esophageal cancer.3.We constructed a Transwell co-culture system to imitate the esophageal cancer microenvironment.EC9706 cells transfected with Cy3-labeled miR-21-5p and miR-93-5p mimics were treated as donor cells while untreated EC9706 cells as recipient cells.The transfer of Cy3-labeled miRNAs between donor and recipient EC9706 cells were observed under a fluorescence microscope,and the transfer efficiency was calculated by qRT-PCR.,EdU assay,Annexin-V FITC&PI assay,transwell assay and flow cytometry were used for the analysis of recipient EC9706 cells proliferation,apoptosis,migration,invasion ability and cell cycle distribution,respectively.The regulating effects of exosome-shuttling miRNAs on their target genes and downstream tumor-related genes were analysis by a qRT-PCR and Western blot.Also,a dual-luciferase reporter assay was used for the direct verification of miR-21-5p and miR-93-5p and their target genes.4.As the previous Transwell co-culture system,EC9706 cells transfected with Cy3-labeled miR-21-5p were treated as donor cells while HUVEC as recipient cells.We also observed the transmission and calculated the transfer efficiency of EC9706 cells derived-exosome-shuttling miR-21-5p to HUVEC cells.EdU assay,transwell assay and blood vessel formation experiment were used for the analysis of HUVEC proliferation,migration,and angiogenesis,respectively.Dual-luciferase reporter assay by constructing the wild type of PTEN 3 'UTR vector was applied to determine the direct com'bination between miR-21-5p and PTEN 3'UTR.QRT-PCR and Western blot were used for the detection of PTEN mRNA and protein in recipient HUVEC cells.The expression level of PTEN downstream tumor-related protein in PI3K/Akt/eNOS/NO pathways were detected by Western blot.Meanwhile,the migration-related MMP2 and MMP9 mRNA level were tested using qRT-PCR.5.Human mononuclear THP-1 cells were induced and differentiated into TAM with the treatment of phorbol myristate acetate(PMA)and human recombinant interleukin(IL)-4.EC9706 cells transfected with labeled miR-21-5p and miR-93-5p mimics were treated as donor cells while the successfully induced TAM were treated as recipient cells through the co-culture system.QRT-PCR were applied for the expression level detection of autophagy-related genes including ATG5,ATG12,BECN1,SQSTMA,LC3A and LC3B.Results:1.Identification and selection of miRNAs from EC9706 cells and their corresponding exosomesPrecipitates isolated from the culture supernatant of EC9706 cells have a circular or elliptic shape that was limited by a lipid bilayer and diameters ranging from 30?60 nm,which were fit to the morphology of exosomes.Western blot analysis confirmed the presence of the known exosomal membrane protein,CD63,which is an exosomal marker in the precipitates.The above results proved that the precipitates were exosomes indeed.The lengths of unique small RNA clean reads in EC9706 cells and exosomes were ranged from 19-24nt,which fit the sequences characters of miRNAs.All unique small RNA clean reads from the small RNA libraries of EC9706 cells and exosomes were compared with the known human miRNAs in miRBase 18.0.We profiled the miRNAs and found 48 known and 12 novel miRNAs in both of cells and exosomes.The expression levels of mature miRNAs in EC9706 cells were significantly higher than exosomes,and among the 12 common novel miRNAs,8 miRNAs were upregulated by more than two-fold in the exosomes.Furthermore,in agreement with the sequence data,qRT-PCR results confirmed that the expression levels of the 5 miRNAs that showed the highest expression in exosomes were relatively similar to those in EC9706 cells.2.Screening of candidate differentially expressed miRNAs in plasma of esophageal cancer patientsMicroarray analysis identified 20 miRNAs that were differentially expressed in the plasma of esophageal cancer patients compared with healthy volunteers.Combined with the Solexa high-throughput sequence results of EC9706 cells and exosomes,we selected miR-21-5p and miR-93-5p as our further research objects after the qRT-PCR verification in a larger population.We have recognized that the dysregulated target genes of miR-21-5p and miR-93-5p could be grouped to 72 and 65 GO classification respectively by bioinformatics analysis.KEGG pathway analysis revealed that the target genes of miR-21-5p and miR-93-5p have dramatically participated multiple pathways related to carcinogenesis such as mTOR?FoxO?PI3K-Akt?TGF-beta signaling pathways.Conditioned logistic regression analysis indicated that the upregulation of miR-21-5p and miR-93-5p in plasma could increase the risk of esophageal cancer.The corresponding area under the receiver operating characteristic curves(AUCs)of miR-21-5p and miR-93-5p were 0.598 and 0.575 while the combined diagnosis value of them was 0.619.Population-based epidemiological data analysis showed that statistically significant distribution of smoking,drinking,and history of gastrointestinal disease were existed between esophageal cancer patients and healthy volunteers,which suggested a correlation with the occurrence of esophageal cancer were existed.3.The regulatory role of exosome-shuttling miRNAs in recipient EC9706 cells3.1 Biological effects of exosome-shuttling miR-21-5p and miR-93-5p in recipient EC9706 cellsAfter the 24h co-cultivation,recipient EC9706 cells were red-fluorescently labeled under a fluorescence microscope.QRT-PCR results showed that the relative expression level of miR-21-5p and miR-93-5p in recipient EC9706 cells were 2.75 and 1.44-fold than miR-negative control(miR-NC)group.Results of biological effects analysis in recipient EC9706 cells showed that both of exosome-shuttling miR-21-5p and miR-93-5p could promote cell proliferation.The transfer of exosome-shuttling miR-21-5p inhibited the early apoptosis but promoted migration and invasion of recipient EC9706 cells.Cell cycle analysis revealed that although there was no significant difference in each phase of recipient EC9706 cells after co-cultured with miR-93-5p mimics transfected donor EC9706 cells,there existed the trend of Gl/S arrest.3.2 Regulatory mechanism of exosome-shuttling miR-21-5p in recipient EC9706 cellsWe have filtered 55 candidate target genes of miR-21-5p using.bioinformatics prediction method.Then PDCD4,which was related to the apoptosis,proliferation,migration and invasion of tumor cells,was selected as the research object.After the 24h cocultivation,qRT-PCR and Western blot results suggested that miR-21-5p secreted by donor EC9706 cells can be effectively uptake by recipient EC9706 cells and regulated PDCD4 expression at a post-transcriptional level in recipient EC9706 cells.Results of dual-luciferase reporter assay indicated that miR-21-5p directly binds to the 3'UTR of PDCD4 genes at "AUAAGCU" region.Compared with the miR-NC group,the mRNA and proteins expression level of MMP2 and MMP9 were increased significantly,which suggested that exosome-shuttling miR-21-5p may play a major role in the JNK downstream signaling pathway involved in tumor migration and invasion.3.3 Regulatory mechanism of exosome-shuttling miR-93-5p in recipient EC9706 cellsPTEN was selected as the candidate target gene of miR-93-5p,and it was found that exosome-shuttling miR-93-5p could markedly inhibit the protein translation of PTEN in recipient EC9706 cells.Results of dual-luciferase reporter assay revealed that miR-93-5p overexpression in EC9706 cells significantly reduced luciferase activity of pmiR-Report-WT-PTEN plasmid,which indicated that miR-93-5p could directly bind to the 3'UTR of PTEN genes.It was found that the protein expression level of cell cycle related genes(p21 and Cyclin D1)were significantly decreased.After the addition of LY294002,the inhibitor of PBK/Akt,the ability of recipient EC9706 cell proliferation was reduced by exosome-shuttling miR-93-5p.The trend of Gl/S arrest was also weaker,and the expression level of p21 and Cyclin D1 protein were restored by 26.71%and 10.33%,respectively.4.The regulatory role of exosome-shuttling miR-21-5p in recipient HUVEC cells4.1 Biological effects of exosome-shuttling miR-21-5p in recipient HUVEC cellsAfter the 24h co-cultivation,recipient HUVEC cells were red-fluorescently labeled under a fluorescence microscope.QRT-PCR results showed that the expression level of miR-21-5p in recipient HUVEC cells was 1.23-fold than the miR-NC group.Results of EdU assay revealed that exosome-shuttling miR-21-5p could promote the proliferation of HUVEC cells.And the transfer of exosome-shuttling miR-21-5p encouraged the migration of HUVEC cells.Blood vessel formation experiment of recipient HUVEC revealed that the total tubule length,number of closed meshes and junctions was 1.12,1.17,1.17-fold than the miR-NC group.4.2 Regulatory mechanism of exosome-shuttling miR-21-5p in recipient HUVEC cellsPTEN,which was closely related to angiogenesis,was selected as the candidate target gene of miR-21-5p in HUVEC cells.Results of dual-luciferase reporter assay revealed that miR-21-5p overexpression in HUVEC cells significantly reduced luciferase activity of pmiR-Report-WT-PTEN plasmid,which indicated that miR-21-5p could directly bind to the 3'UTR of PTEN genes.After the 24h cocultivation,qRT-PCR and western blot results suggested that miR-21-5p secreted by donor EC9706 cells could regulate PTEN expression at a post-transcriptional level in recipient HUVEC cells.It was found that phospho-Akt(p-Akt)(Ser473),one of the downstream genes of PTEN,was significantly increased by 14.18%in recipient HUVEC cells.Meanwhile,phosphor-eNOS(p-eNOS)(Ser1177),the downstream gene of p-Akt,was significantly decreased by 42.13%.After the addition of LY294002,the expression level of p-Akt(Ser473)and p-eNOS(Ser1177)were restored partially.However,after the addition of L-NAME,the inhibitor of eNOS,the expression level of p-Akt(Ser473)was not significantly changed but restored by 28.08%for p-eNOS(Ser1177).Compared with the miR-NC group,the expression level of phospho-Erkl/2(p-Erk1/2)(Thr202/Tyr204),one of the proliferation-related proteins in downstream of NO,was decreased significantly.The mRNA expression level of MMP9 also showed a decreasing trend.5.A preliminary exploration of exosome-shuttling miRNAs in regulating the autophagy of recipient TAMsAfter treatment of PMA in THP-1 cells for 48h,THP-1 cells were induced from 'grape string-shape' suspended growth to adherent growth.Continued with the treatment of recombinant IL-4 for 48h,THP-1 cells showed irregular shapes,some extended pseudopodia and the connection between cells formed,which were fit to the morphology of TAMs.After the 24h co-cultivation,qRT-PCR results showed that the mRNA expression level of ATG12 and BECNl in miR-21-5p mimics transfected-recipient TAMs were 1.26 and 1.63-fold respectively and BECN1 mRNA in miR-93-5p mimics transfected-recipient TAMs was 1.34-fold than the miR-NC group.There was no significantly difference detected in the other autophagy-related genes(ATG5,ATG12,BECN1,SQSTMA,LC3A,and LC3B).Conclusions:1.We have successfully isolated exosomes from the culture supernatant of esophageal cancer cells EC9706.It was discovered that there were 342 and 48 known mature miRNAs in EC9706 cells and corresponding exosomes,respectively,which existed a higher expression level in cells than exosomes.Furthermore,12 novel miRNAs were found in both of cells and exosomes,including 8 up-regulated miRNAs and 4 down-regulated miRNAs.2.Microarray analysis identified 20 miRNAs that were differently expressed in the plasma of esophageal cancer patients compared with healthy volunteers.MiR-21-5p and miR-93-5p were identified up-regulated in the plasma from esophageal cancer patients after the qRT-PCR verification in a larger population.It was revealed that the upregulation of miR-21-5p and miR-93-5p in the plasma samples could increase the risk of esophageal cancer.Both of them could distinguish esophageal cancer patients from healthy volunteers.However,both of the sensitivity and specificity of these two miRNAs were not high enough;it is imperative to expand the plasma samples for further study.3.In the microenvironment of esophageal cancer,EC9706 cells-derived exosome-shuttling miR-21-5p may participate the proliferation,apoptosis,migration,invasion of recipient EC9706 cells by targeting PDCD4.Exosome-shuttling miR-93-5p may take part the proliferation and cycle distribution of recipient EC9706 cells through PTEN/PI3K/Akt signaling pathway.4.Exosome-shuttling miR-21-5p released by EC9706 cells may regulate the proliferation,migration,and angiogenesis of recipient HUVEC cells by activating PTEN/Akt/eNOS/NO signal pathway and affecting the expression of downstream migration-related genes.5.Exosome-shuttling miR-21-5p and miR-93-5p derived from EC9706 cells may regulate the autophagy level of recipient TAMs by changing the expression of autophagy-related genes(ATG12 and BECN1),which suggested that they may play a significant role in cell-cell communication in the microenvironment of esophageal cancer.