The Mechanism Of Renal Carcer Cell Derived Exosomes On The Invasion And Metastasis Of Renal Cancer Cell Line786-0

ObjectiveTo extract exosomes from the renal cancer cell line786-0supernant, toinvestigate the effect of786-0derived exosomes on the invasion andmetastasis of786-0and the possible mechanism; To isolate and purify theexosomes from renal cancer and health volunteers serum, then Itraq tabbedflight mass spectrometry derived differential protemics were processed tofind the invasion related proteins; siRNA were used to selectively preventthe renal caner invasion related protein Thrombospondin-1expression inrenal cancer cell line secreted exosomes, then to further investigate thepossible role of renal caner secreted Thrombospondin-1in the invasion andmetastasis of renal cancer cell line786-0and the possible mechanism. Wehope that the above research will provide a theoretical supplement for renalcancer metastasis and new ideas biological treatment of metastatic renal cellcarcinoma.Methods1.Isolation and identification of exosomes from786-0supernatant:Ultracentrifugation and sucrose/water density gradient ultracentrifugation were used to isolate and purified the786-0derived exosomes, Transmissionelectron microscope(TEM) were used to observe the morphology of isolatedexosomes, Western blot were used to analyze the expression of HSP70,ICAM-1and G250. Bradford method were used to quantitatively Analyzethe total protein content of exosomes.2.The effect of renal cancer cell line786-0derived exosomes on theinvasion and migration of renal carcinoma cell line786-0: exosomes (100μg/mL), a specific inhibitor of exosomes Amiloride,5-(N, N-Dimethyl)-,hydrochloride,(7mmol/l) were co-cultured with786-0cells, after24hours,the786-0cells were collected. The wound healing assay, Matrigel basedinvasive Transwell cell experiment and cell adhesion experiments wereprocessed to check the invasion and migration capability of786-0cells.3.Western-blot were used to detecte the expression changes of CXCR4and matrix MMP-9on786-0cell.4.Isolation of exosomes from serum: the serum of18cases renal cancerpatients and6heaith volunteers were collected, Ultracentrifugation andsucrose/water density gradient ultracentrifugation were used to isolate andpurified the exosomes.5.Protemics analysis: Exosomes proteomic analysis were processed byisobaric tags for relative and absolute quantification (iTRAQ) techniquecombined with LC-MALDI-TOF/TOF.6.Protemics results analysis: Protemics results were analyzed by KEGG and PUBMED. The invasion related proteins were selected.Immunohistochemical technique were used to detect theThrombospondin(TSP-1) expression of26cases of renal cell carcinomaspecimens and26cases of cancer adjacent tissues. Western-blot were usedto detected the TSP-1expression in18cases of renal cell carcinoma patientsand6healthy volunteers.7.TSP-1knockdowm in exosomes. TSP-1interference fragment wereproduced by chemical synthesis, then was transfected into786-0.Flow cytometry were used to detect transfection efficiency. Stabletransfected cell line were collected. Exosomes extracted from ransfected celllines culture supernatant were harvested. Western-blott were used to analyzethe TSP-1expression.8.The pivotal role of secreted TSP-1in renal cancer cell line786-0andthe possible mechanism. The TSP-KO exosomes, and normal exosomeswere co-cultured with normal786-0cells for24hours. wound healing assay,Matrigel based invasive Transwell cell experiment and cell adhesionexperiments were processed to check the invasion and migration capabilityof786-0cells. Western-blot were used to detecte the expression changes ofCXCR4and matrix MMP-9on786-0cell.Results1.exosomes were successfully extracted from786-0cell culturesupernatant and patients or healthy volunteer serum. The morphology of the 786-0derived exosomes under Transmission electron microscopy werespheroid. Western blot found renal cell carcinoma cell line786-0inducedexosomes express intercellular adhesion molecule (ICAM-1), heat shockprotein70(HSP70) and G250. The Bradford methods found that theconcentration of extracted exosomes were1.5mg/ml～2.0mg/ml. Theabove findings lay the foundation for the following experiments.2.the renal cancer cell line786-0cells co-cultured with exosomesshowed higher cell migration capability than that of exosomes inhibitiongroup and PBS group, the number of migrated cells per unit area were55.84±7.60;16.06±4.08;29.17±1.72; respectively; cell invasion capabilitysignificantly increased, the cell number of traveling through the MatrigelTranswell basement membrane after24hours as were87.5±7.8,29.3±11.7,57.6±5.4,respectively; cell adhesion capability were significantlydecreased, the number of adherent cells in three groups of experiments were42.5±6.5,71.5±7.5,51.5±8.5, respectively. Western-blott were used todetect the CXCR4and MMP-9expression in the three groups. The CXCR4and MMP-9expression were significantly increased in exosomes treatmentgroup.3.ITAQ analysis showed that the exosomes containing393proteins, inwhich51proteins were highly expressed in renal cell carcinoma patientderived exosomes and5proteins were lowly expressed in renal cellcarcinoma patient derived exosomes. There exist three invasion related functional proteins among the highly expressed protein:Thrombospondin(TSP-1), Extracellular matrix protein1(ECM-1), Pigmentepithelium derived factor (PEDF). TSP-1were with the highest expressionamong the invasion related proteins(P114:113=4.092). Among the lowlyexpressed proteins, Insulin like growth factor binding protein were relatedwith the renal cancer invasion.4.Immunohistochemistry confirmed that the expression of TSP-1inrenal cancer tissue and in adjacent tissue were with no significantlydifference. However, Western-blot found that TSP-1expression in renalcancer patients serum were significantly higher than that in healthyvolunteers.5.Flow cytometry showed that TSP-1interference fragmentsuccessfully transfected into786-0cells by siRNA technology. Thetransfection rate were46.21%. The expression of TSP-1in786-0derivedexosomes were decreased when786-0cell transfected with TSP-1interference fragment.6.TSP-1protein Silenced exosomes decreased the migration ability of786-0cells compared with normal exosomes, the number of cells per unitarea were16.44±6.08,53.84±6.70, respectively; cell invasion capabilitywere significantly decreased, the cell number of traveling through theMatrigel Transwell basement membrane after24hours as were22.5±12.8,89.5±7.2, respectively; cell adhesion ability were enhanced, the number of adherent cells were67.5±6.5,38.5±6.5, respectively. Western-blott wereused to detect the CXCR4and MMP-9expression in the two groups. TheCXCR4and MMP-9expression were significantly decreased in TSP-1KOtreatment group.Conclusions1.The786-0derived exosomes and renal cancer derived exosomescould be successfully extracted and purified from the supernatant of renalcancer cell line786-0and serums by the use of ultrafiltration and sucrose/water density gradient ultracentrifugation. The exosomes expressed ICAM-1,HSP70, G250could be used for the follow-up co-culture experiments. Theseexperiments laid a solid basis for further research.2.Renal cell carcinoma cell line786-0derived exosomes can promotethe migration and invasion of786-0cells, reduce the cell adhesion ability.The above function could be prevented by exosomes specific inhibitorAmiloride,5-(N, N-Dimethyl)-, hydrochloride. The possible mechanismfor renal carcinoma cell line786-0derived exosomes promoting migrationand invasion of786-0cells might be that786-0derived exosomes couldenhance the786-0cell CXCR4and MMP-9expression. These findings mayprovide experimental evidence for the following experiment.3.Itraq technology could be used to analyse the differential protein ofexosomes in the serum of renal cancer patients and healthy volunteers. Theresults showed that TSP-1were highly expressed in renal cancer patients serum and highly related with renal cancer invasion. High expression ofthrombospondin-1may be a specific molecular target for renal cellcarcinoma invasion. These findings may provide a research area forexosomes derived renal cancer cell invasion.4.Immunohistochemistry confirmed that the expression of TSP-1inrenal cancer tissue and in adjacent tissue were with no significantlydifference. However, Western-blot found that TSP-1expression in renalcancer patients serum were significantly higher than that in healthyvolunteers. These findings suggested that TSP-1in tumor secreted exosomesmy play a pivotal role in renal cancer invasion and may provide a theoryevidence and experimental basis for the following experiment.5.TSP-1interference fragment successfully transfected into786-0cellsby siRNA technology. The expression of TSP-1in786-0derived exosomeswere decreased when786-0cell transfected with TSP-1interferencefragment.TSP-1silenced exosomes derived from786-0can inhibitthe migration and invasion of786-0cells, enhance the adhesion ability. Thepossible mechanism might be TSP-1silenced exosomes can inhibit theexpression of CXCR4and MMP-9.