| Research BackgroundRenal cell carcinoma (RCC) is a common malignant tumor of theurinary system, of which the incidence is second only to the bladder cancer,showing an increasing trend. More than50%of the patients have no specificsymptoms. And the RCC often have entered into middle-late phrase whenthey are accidentally discovered. About one third of the patients had cancermetastasis, which prompt a poor prognosis. The RCC is treated mainly bysurgical operation in present, but early diagnosis rate is low. Also theradiotherapy and chemotherapy are not sensitive, and the immune therapy oflong-dated effect is limited, therefore the overall treatment effect is still poor.In order to improve the early diagnostic rate and the curative effect of theRCC, it is very necessary to strengthen the basic research of RCC to clarifyits development mechanism.Exosomes is a30-100nm membranous capsule released by the fusion of multivesicular bodies and the cell membrane of eukaryotic cells, which iswidely distributed in cell microenvironment around with complexcomposition and a variety of proteins. Studies have shown that exosomeswas closely related to tumor immune regulation. The RCC-derivedexosomes was extracted successfully in the preliminary work by our studyteam for the first time. It was found that the specific RCC antigen G250,HSP70and intercellular adhesion molecules (ICAM-1) were rich inexosomes, which could reflect the biological information of RCC protein,and can also induce mononuclear cells differentiate into PD-L1+myeloid-derived suppressor cells. Therefore, RCC-derived exosomes couldaffect the incidence and development of RCC, as well as the immune effect.The RCC-derived exosomes widely exists in tumor cell periphery. Burthe following questions including the mechanisms are still not clear: Does ithave an effect on the biological behaviors such as the growth, proliferation,apoptosis, invasion and migration of the RCC or not? If there is an impact, isit promoting effect or inhibition effect? These question have not beenreported at present. Can the immunity regulation of tumormicroenvironment enhance the immunity or induce immune escaping?Under what circumstances, it can enhance the immunity or induce immuneescaping? Therefore, the effect of the RCC-derived exosomes on thebiological behavior of RCC and the direct and indirect effect of exosomes,and the impact on the local and whole body, to comprehensively explore the effect of RCC-derived exosomes on the development of RCC. And themechanisms of incidence, development and immune escaping mechanism ofRCC will be revealed through the study to provide new ideas forimmunotherapy of RCC.ObjectiveRenal cancer786-0cells were taked as the experimental object toextract the exosomes in this study. the potential effects of exosomes derivedfrom renal cancer cells on the proliferation,apoptosis,invasion and migrationof renal cancer own cells, and the apoptosis of human Jurkat T cells wereinvestigated in vitro. At the same time, the corresponding mechanism wereexplored preliminarily in the hope of revealing the roles of exosomes derivedfrom renal cancer cells in the malignant development of renal cell cancer andproviding new ideas for immunotherapy of renal cancer.MethodsExosomes were purified from786-0cells by sequential centrifugationsand ultrafiltrations、ExoQuick exosomes precipitation solution respectively.1．Exosomes were characterized by transmission electron microscope(TEM).2．Quantitative analysis of total protein of exosomes were determinedby Bradford method.3．The effects of the exosomes on the proliferation of renal cancer celllines and Jurkat T cells were analyzed with the Cell Counting Kit-8(CCK-8) assay.4．Flow cytometry with Annexin V-FITC/PI double staining was usedto detect the cells apoptosis.5．After the Wright-Giemsa staining, the changes of morphology ofJurkat T cells were observed under optical microscope.6．Secretion functions of Jurkat T cell were detected by ELISA assay.7．Effects of exosomes on apoptosis of Jurkat T cells were detected bysoluble Fas block experiment after using soluble Fas block.8．The gene expression was detected by RT-PCR.9．The protein expression level was detected by Western blotting.10．Statistical methods: T test was used by software SPSS17.0.Results1． The ultrafiltration, heavy-water sucrose density gradientultracentrifugation, and ExoQuick exosomes kit were respectively used tosuccessfully extract the786-0RCC-derived exosomes supernatant fluid forRCC. The morphology of the exosomes obtained by the two methods weresimilar and the total amount of proteins were equivalent. Also, thecompositions of the characteristic proteins were the same. The lipid bilayermembrane disk structures were all observed in exosomes under thetransmission electron microscope, with the diameter of about30-l00nm. Theprotein concentrations extracted were between1500~2000ug/mL of.Western blotting analysis showed that the protein compositions of exosomes extracted by two methods mainly distributed from35to66kd. The RCCspecific antigen G250, HSP90, CD63, TSG101and other molecules wererich on exosomes surface. But in the practical application, the two methodshad their own advantages and disadvantages.2．The786-0RCC-derived exosomes could promote proliferation of786-0and ACHN cells, in a time and dose dependent manner. After the786-0cells were treated with200ug/mL of and400ug/mL of exosomes, thecell proliferation rates were respectively124.09%and141.67%that of thecontrol group; while after72h, cell proliferation rates were respectively169.97%and174.4%that of the control group. After ACHN cells weretreated with200ug/mL of and400ug/mL of exosomes for24h, cellsproliferation rates were respectively126.36%and140.73%that of thecontrol group; while after72h, cell proliferation rates were respectively167.34%and177.11%that of the control group. The786-0RCC-derivedexosomes could inhibit the apoptosis of786-0and ACHN cells, in a doseand time dependent manner. After786-0cells were treated with10ug/mL ofexosomes for24h, the apoptosis rate was （3.76±0.12）%; while after72h,the apoptosis rate was （3.37±0.20）%; After treated with400ug/mL ofexosomes for24h, the apoptosis rate was （0.94±0.06）%; while after72h,the apoptosis rate was only （0.37±0.05）%. After treated with10ug/mL ofE for24h, the apoptosis rate of ACHN cells was （3.83±0.10）%; and after72h, the apoptosis rate was（3.55±0.23）%; after treated with400ug/mL of exosomes for24h, the apoptosis rate was （0.95±0.04）%, while after72h,the apoptosis rate was only （0.38±0.03）%.3．The786-0RCC-derived exosomes could promote the cell migration.In the invasion experiment, after786-0cells were treated with100ug/mL ofexosomes for72h, the number of invasive cells that penetrated through theMat glue Transwell little room basement membranes was （147.5±7.87）;and after treated with400ug/mL of exosomes for72h, the number ofinvasive cells was （270.33±8.48）. In the migration experiment, after786-0cells were treated with100ug/mL of exosomes for72h, the number ofmigrated cells across the membrane in experimental group was（168.1±5.56）, and after treated with the400ug/mLof exosomes for72h,the number of migrated cells increased to （293.67±7.89）.4．The expression of cyclinD1, caspase-3, Bcl-2and Bax mRNA of786-0RCC treated by different concentrations of exosomes (100,400ug/mLof) for24h were detected by RT-PCR method. Results showed that comparedwith the control group, the expression of cyclinD1and Bcl-2mRNAincreased, while the expression of Bax mRNA decreased, in a dosedependent manner. But the expression of caspase-3mRNA had no obviouschange. Compared with the control group, the expression of cyclinD1andBcl-2were found to increase gradually with the increase of the concentrationof E through the Western blotting analysis, while the expression of Bax andcaspase-3protein gradually reduced with the increase of the concentration of exosomes. At the same time, the expression levels of p-Aktand p-ERK1/2proteins were detected, and the results suggested that exosomes couldup-regulate the expression of p-Akt and p-ERK1/2in a dose dependentmanner. In the cell migration, the expression of MMP-2, MT1-MMP andNF-κB proteins were analyzed with Western blotting. And the resultsshowed that compared with the control group, the expression of MMP-2,MT1-MMP and the NF-κB proteins increased with the increase of theconcentration of exosomes.5．The786-0RCC-derived exosomes could inhibit the growth of T cells.After the T cells were treated with10ug/mL of exosomes for24h, thegrowth inhibition rate was （19.64±0.92）%, while it was （36.24±1.12）%after72h; and after it was treated with400ug/mL of exosomes for24h,the growth inhibition rate was （55.96±1.35）%, while it was （76.51±1.37）%after72h. exosomes could induce T cell apoptosis. After T cells weretreated with10mg/mL of exosomes for8h, the apoptosis rate was（7.31±1.32）%,whileitwas（20.19±1.47）%after24h;afteritwastreatedwith400mg/mL of exosomes for8h, the apoptosis rate was （27.28±1.29）%, while it was （41.72±0.88）%after24h. At the same time, themorphological changes of T cell were detected by the r’s-JiM stainingmethods, and the results showed that the volume of apoptosis T cell shrankand the cytoplasm gradually concentrated. And the nuclei cracked intofragments and the apoptotic body was produced. 6．Exosomes could significantly inhibit the secretion of IL-2, IFN-γ,IL-6and IL-10of T cells. The functions of secreting cytokines of Jurkat Tcells after they were treated with400ug/mL of exosomes for8h weredetected by ELISA method, and the results showed that the functionsdecreased significantly. And the concentrations of IL-2, IFN-γ, IL-6andIL-10decreased to (59.2±14.6),(33.1±4.55),(62.8±16.1)and (33.7±14.3)respectively.7．The expression of FasL protein was high in the exosomes. Theapoptosis rate changes of T cells were detected by the soluble Fas blockingexperiment and the results showed that the apoptosis of T cells at8,16, and24h significantly decreased and the effect of exosomes on Jurkat T cellapoptosis was almost reversed.8．After Jurkat T cells were treated with different concentrations ofRCC-derived exosomes for24h, the expression of crackedcaspase-3,caspase-8,caspase-9fragments were found to increase in a dosedependent manner; at the same time, the expression level of Bax proteinincreased and the expression of Bcl2protein decreased.Conclusions1． The extracting methods of ultrafiltration, heavy-water sucrosedensity gradient ultracentrifugation and ExoQuick kit for exosomes werefeasible and reliable, but they all had advantages and disadvantages. The786-0RCC-derived exosomes was a nanoscale membranous capsule, containing the important information of786-0, and expressed the RCCspecific antigen G250,HSP90,CD63,TSG101.2．The786-0RCC-derived exosomes could promote proliferation of786-0RCC and ACHN cells, while inhibit cell apoptosis at the same time ina time and dose dependent manner. The786-0RCC-derived exosomes couldpromote the cell migration, thus promote the malignant development of renalcancer cells directly.3．The increased expression of cyclinD1molecules, the decreasedexpression of caspase-3protein, the decreased ratio of Bax/Bcl-2and theactivation of ERK and Akt signaling pathways might be the molecularmechanisms of exosomes promoting proliferation and inhibiting theapoptosis.Exosomes could activate MMPs by up-regulating the expressionof NF-κB to degenerate the extracellular matrix for the promotion of RCCmigration.4．The RCC-derived exosomes could induce Jurkat T cell apoptosis ina dose and time dependent manner and inhibit its growth. It promptsexosomes can lower the body’s immune ability, promote the malignantdevelopment of renal cancer cells indirectly.5．The exosomes could significantly inhibit the secretion of IL-2, IFN-γ,IL-6and IL-10of Jurkat T cells.6．The effect of the RCC-derived exosomes on inducing Jurkat T cellapoptosis could be reversed by soluble FAS experiment. 7． Fas/FasL,Caspase-3,Caspase-8,Caspase-9and the Bcl-2proteinfamily might participate in the inducing effect of the RCC-derived exosomeson the cell apoptosis. |
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