| The metastasis of Prostate cancer(PC)has always been a difficult and serious problem in clinical practice and basic research.Previous studies have shown that RelB,a key molecule in the non-canonical NF-κB(Nuclear factor κ-light-chain-enhancer of activated B cells)signaling pathway,is involved in the occurrence,progression,invasion and metastasis of PC.The unique tumor microenvironment(TME)can accelerate the occurrence of PC metastasis.However,the role of RelB in the three-dimensional structure of TME of PC has not been reported.In addition to tumor cells,there are a large number of stromal fibroblasts and exosomes secreted by cells in the TME of PC.Previous studies have reported that fibroblasts can transform into carcinogenic associated fibroblasts(CAFs)after being activated in the TME.CAFs have high secretory activity and influence the occurrence and development of tumors by regulating cell metabolism,epithelial-mesenchymal transformation,neovascularization,infiltration and metastasis of stem cells,and immunosuppression.Exosomes,secreted by normal cells or tumor cells,regulate the carcinogenesis by mediating the transfer of nucleic acids,proteins,lipids and other active substances.It has been shown that there is mutual regulation between PC cells and prostate fibroblasts in the TME through the co-culturing of these two kinds of cells.The expression of RelB affected the two-way regulation processes.Proteomic analysis showed that RelB regulated the expression of exosomal proteins in PC cells.The role of Intercellular adhesion molecule(ICAM1),a key target of RelB,in the metastasis TME of PC has not been reported.The aim of this study is to investigate the interaction among PC cells,fibroblasts and exosomes in the TME of PC.By studying the impact of exosomes carrying ICAM1 on PC cells and fibroblasts in the TME of PC,the biological functions and mechanism of RelBmediated TME in the metastasis process of PC were clarified.Part Ⅰ The role and mechanisms of RelB in the mutual regulation between prostate cancer cells and fibroblastsAim To clarify the role and mechanism of RelB in the mutual regulation between PC cells and fibroblasts,by analyzing the effects of cell-cell indirect or direct co-culture on the biological behaviors,and the protein expression of NF-κB signal pathway of tumor cells and fibroblasts.Methods A prostatic fibroblast cell line carrying puromycin resistance and GFP labeling based on WPMY-1 cell was established by virus infection.In the indirect cell coculture model,WPMY-1-GFP cells were cultured by the conditional medium(CM)collected from the control cells(sictrl)and RelB-knockdown(siRelB)PC cells.Otherwise,sictrl and siRelB cells were cultured by the CM collected from WPMY-1-GFP cells,respectively.The cell growth,migration and invasion abilities of WPMY-1-GFP cells,sictrl and siRelB cells after indirect co-culture were dynamically examined by a real-time xCELLigence system.The cellular proliferation and apoptosis after indirect co-culture were detected by CCK-8 assay and AnnexinV/PI double staining.The expression of markers of fibroblast and CAFs in WPMY-1-GFP cells were detected by immunohistochemistry(IHC).The secretion of soluble factor of WPMY-1-GFP cells,sictrl and siRelB cells were detected by ELISA assay.The expression of NF-κB signaling pathway proteins and metastasis-related proteins in WPMY-1-GFP cells,sictrl and siRelB cells after co-culture were detected by western blot.The direct cell-cell co-culture model was established by mixed sictrl or siRelB cells with WPMY-1-GFP cells at a ratio of 1:1.The changes in the above biological characteristics of WPMY-1-GFP cells were analyzed after removing PC cells upon puromycin treatment.The changes in the above biological characteristics of sictrl or siRelB cells were analyzed after removing WPMY-1-GFP cells upon G418 treatment.Another co-culture model was established by mixed sictrl or siRelB cells with HUEVC-GFP cells at a ratio of 1:1.The changes in the proliferation,migration and invasion abilities of sictrl or siRelB cells were analyzed after removing HUEVC-GFP cells upon G418 treatment.Results A WPMY-1-GFP cell line with puromycin-resistance was successfully established.Under the indirect co-culture with PC cells,the cell growth,proliferation and secretion of soluble factors of WPMY-1-GFP cells were significantly promoted,the apoptosis of WPMY-1-GFP cells were inhibited.The CAFs markers,Desmin and FAP,in WPMY-1-GFP cells were increased after co-culture.The protein expression of RelB and MMP2 in WPMY-1-GFP cells were up-regulated.The effect of siRelB cells were weaker than those of sictrl cells during the above processes.Under the indirect co-culture with fibroblasts,the cell growth,proliferation,migration,invasion abilities and secretion of soluble factors of PC cells were significantly inhibited.The apoptosis of PC cells were promoted.The expression of RelB protein in the nucleus of the PC cells were significantly down-regulated.The expression of metastasis-related proteins MMP9,uPA and Integrin β1 were also down-regulated.The effect of fibroblasts on sictrl cells was stronger than that on siRelB cells during the above processes.After the direct co-culture with sictrl cells,the cell growth,proliferation and apoptosis of WPMY-1-GFP cells were not significantly affected.The secretion capacity of soluble factors was enhanced.The CAFs markers Desmin and FAP were induced.The protein expression of RelB in the cytoplasm and nucleus were significantly up-regulated.Under the direct co-culture with siRelB cells,the cell growth and proliferation of WPMY-1-GFP cells were significantly reduced.The apoptosis of WPMY-1GFP cells was increased.Only the secretion of CXCL13 was increased.CAFs markers were negatively expressed.Co-culture with fibroblasts significantly inhibited the cell growth,proliferation,migration and invasion abilities of PC cells.The apoptosis of PC cells was promoted.The expression of NF-κB signaling proteins and metastasis-related proteins MMP9,uPA and Integrin β1 in sictrl and siRelB cells were also down-regulated.The effect of fibroblasts on sictrl cells was stronger than that on siRelB cells during the above processes.Co-culture with HUEVC-GFP did not influence the proliferation,migration and invasion abilities of PC cells.Conclusions1.RelB significantly promotes the cell growth and proliferation,inhibits the apoptosis,induces the transformation into CAFs,and enhances the secretory activity of fibroblasts through indirect co-culture.RelB can maintain the survival of fibroblasts,inhibits the apoptosis,promotes the transformation into CAFs and enhances the secretory activity of fibroblasts through direct co-culture.2.Prostate fibroblasts can inhibit the cell growth and proliferation of PC cells,promote their apoptosis and reduce the secretion of soluble factors through indirect or direct coculture,which may be related to the down-regulation of NF-κB signaling proteins in cancer cells.At the same time,fibroblasts inhibited the migration and invasion abilities of tumor cells,which may result from the down-regulated expression of metastasis-related proteins.Prostate fibroblasts plays more significant roles in the regulation of PC cells expressing higher RelB.3.RelB expression is involved in the bidirectional regulation between tumor cells and fibroblasts in the microenvironment of PC.Part Ⅱ RelB regulates the proteins panel in exosomes derived from prostate cancer cellsAim To elucidate the role of RelB in regulating the proteins expression profile of exosomes derived from PC cells,and to identify candidate target proteins.Methods Exosomes were extracted by ultra-centrifugation.The morphology of exosomes were observed by transmission electron microscopy(TEM).The particle size of exosomes was analyzed by Nanoparticle tracking analysis(NAT)technique.The expression of exosome specific proteins CD63,CD81 and Flotillin were detected by western blot.Liquid chromatograph-mass spectrometry was used to detect exosomal proteins.Proteomic analysis was performed based on bioinformatics tools.The expression of exosomal proteins were compared between sictrl and siRelB cells.The differentially expressed proteins(DEPs)were analyzed by heat map,Venn diagram,GO enrichment and KEGG analysis.The correlation between the RelB gene and the ICAM1 gene was explored by UALCAN website.The protein-protein interaction between RelB and ICAM1 was analyzed by GeneMANIA.The expression of RelB and ICAM1 in PC patients were detected by IHC.Their clinical significance were analyzed.The relationship between RelB and ICAM1 was explored by dual-luciferase report assay.The expression of ICAM1 in exosomes derived from sictrl and siRelB cells were verified by western blot.ICAM1-overexpressed PC cell line and control cell line(named hICAM 1 and hctrl)were established by plasmid transfection.The expression of ICAM1 at mRNA and protein levels were detected by qRT-PCR and western blot,respectively.The cell growth,migration and invasion abilities of hICAM land hctrl cells were dynamically observed by a real-time xCELLigence system.The proliferation ability of cells was detected by CCK-8 assay.The apoptosis of cells was detected by AnnexinV/PI double staining.The mRNA and protein expression of ICAM1 in exosomes derived from sictrl,siRelB,hICAM1 and hctrl cells were detected by qRT-PCR and western blot,respectively.The cytomembrane expression of ICAM1 was analyzed by flow cytometry.Results The extracellular vesicles were "cup"-shaped with concave by TEM.The diameter of extracellular vesicles were 30-200 nm by NAT analysis.The protein extracted from extracellular vesicles were positive for CD63,CD81 and Flotillin by western blot.1,259 proteins were detected in exosomes derived from PC cells,of which 1,125 were recorded by exosome database Exocarta,indicating a purity as 89.4%.105 proteins were expressed only in the sictrl cells.160 proteins were expressed merely in siRelB cells.994 proteins were commonly expressed in sictrl and siRelB cells.A total of 192 DEPs were obtained.Compared with the sictrl cells,137 proteins were up-regulated and 55 proteins were down-regulated in exosomes derived from the siRelB cells.The GO enrichment and KEGG pathway analysis suggested that some DEPs mainly played the functions of cell-cell adhesion and cell adhesion,and mainly involved in antigen processing and presentation,local adhesion,and EB virus infection processes.ICAM1 was found to be regulated by NFκB signaling pathway during Epstein-Barr virus infection.ICAM1 was downregulated in the absence of RelB,with an FC of 2.136,and a P<0.001.UALCAN showed that the RelB gene was positively correlated with the ICAM1 gene(Pearson correlation coefficient was 0.71).RelB and ICAM1 expression were significantly higher in tumor tissues when compared with tumor-adjacent tissue samples(P=9.0237E-03 and 9.9639E-04).Genemania showed that RelB and ICAM1 mainly interacted in a co-expression mode.RelB expression was positively correlated with lymph node metastasis and clinical stage in the tissue microarray of PC patients(P=0.023 and 0.019).ICAM1 expression was positively correlated with lymph node metastasis and clinical stage(P=0.017 and 0.035).Compared with tumoradjacent tissue samples,the proportion of samples with high expression of RelB and IC AM1 in tumor group were significantly higher(both P<0.05).There was a positive correlation between RelB and ICAM1 protein expression in the tissue microarray of PC patients(Spearman r=0.406).RelB regulated ICAM1 by binding directly to its promoter.The results of qRT-PCR and western blot showed that the mRNA expression and protein level of ICAM1 after transfection were significantly up-regulated compared with control cells.ICAM1 overexpression significantly promoted the cell growth,proliferation,migration and invasion abilities of PC cells by cell biological analysis.The protein expression of uPA and Integrinβ1 were up-regulated.The mRNA and protein expression of ICAM1 were the highest in the exosomes of hICAM1 cells,followed by sictrl and hctrl cells,and the lowest in the siRelB cells.ICAM1 overexpression did not up-regulate the cytomembrane expression of CD54/ICAM1.Conclusions1.RelB regulates the protein profile in exosomes derived from PC cells.ICAM1 is targeted by RelB.Exosomal ICAM1 is a potential downstream target of RelB.2.The expression of ICAM1 is positively correlated with the cell growth,proliferation,migration and invasion abilities of PC cells.ICAM1 plays a carcinogenic role in PC cells.3.The expression of ICAM1 is significantly positively correlated with lymph node metastasis and clinical stage of PC,suggesting that the high expression of ICAM1 is associated with the progression of PC.4.The PC cells with higher expression of ICAM1 also has higher ICAM1 protein expression in the exosomes,but lower ICAM1 expression on the cytomembrane.Part III The role and mechanism of RelB in prostate cancer metastasis by upregulating exosomal ICAM1Aim To investigate the role and mechanism of RelB in the invasion and metastasis of PC by up-regulating exosomal ICAM1,through co-culture of PC cells or fibroblasts with exosomes.Methods Exosomes were extracted by ultra-centrifugation from the CM of sictrl,siRelB and hICAM1 cells.WPMY-1-GFP cells were co-cultured by hICAM1-exo,sictrl-exo and siRelB-exo,respectively.siRelB cells were co-cultured by hICAM1-exo and sictrl-exo respectively.sictrl cells were co-cultured by hICAM1-exo and siRelB-exo,respectively.The exosomes were labeled with PKH 26.The uptake of exosomes by recipient cells was observed under fluorescence microscope.The uptake rate of exosomes was detected by flow cytometry.The cell growth,migration and invasion abilities of sictrl,siRelB cells and WPMY-1-GFP cells after co-culture with exosomes were dynamically detected by a realtime xCELLigence system.The cell proliferation and apoptosis of sictrl,siRelB cells and WPMY-1-GFP cells after co-culture with exosomes were detected by CCK-8 reagent and AnnexinV/PI double staining.The expression of CAFs markers in WPMY-1-GFP cells were detected by IHC.The secretion of soluble factor in sictrl,siRelB cells and WPMY-1-GFP cells were detected by ELISA assay.The expression of NF-κB signaling pathway proteins and metastasis-related proteins in WPMY-1-GFP cells or sictrl and siRelB cells after coculture with exosomes were detected by western blot.Results Exosomes labeled by PKH 26 were located on the membrane margin or inside the cytoplasm of the recipient cells under the fluorescence microscope.Exosomes were uptaked by more than 90%of the cells by flow cytometry.Co-culture with hICAMl-exo,sictrl-exo and siRelB-exo could promote the cell growth and proliferation,and inhibit the apoptosis of WPMY-1-GFP cells.IHC results showed that co-culture with hICAMl-exo and sictrl-exo induced the positive expression of CAFs markers,Desmin and FAP,in WPMY-1GFP cells,while siRelB-exo only induced the weakly expression of Desmin in WPMY-1GFP cells.Co-culture with the three kinds of cancer cell exosomes enhanced the secretion of IL-6,IL-8,TGF-β1 and CXCL13 to varying degrees in WPMY-1-GFP cells.The protein expression of RelB in the cytoplasm and nucleus of WPMY-1-GFP cells were up-regulated when treated by hICAM1-exo and sictrl-exo.The expression of MMP2 in WPMY-1-GFP cells was significantly up-regulated when co-cultured with hICAM1-exo and sictrl-exo.Coculture with hICAM1-exo and sictrl-exo significantly promote the proliferation,migration and invasion abilities,inhibit the apoptosis of siRelB cells.The promotion and inhibition effects of hICAM1-exo were stronger than that of sictrl-exo.hICAM1-exo also promoted the cell growth of siRelB cells.Co-culture with hICAM1-exo upregulated the expression of ICAM1,Integrin β1,MMP9 and uPA proteins,and enhanced the secretion of IL-6,IL-8,TGF-β1 and CXCL13 in siRelB cells.Co-culture with sictrl-exo upregulated the expression of MMP9 protein,and enhanced the secretion of IL-6,IL-8 and TGF-β1 in siRelB cells.The level of RelB and p52 proteins in the nucleus of siRelB cells were upregulated when treated by both hICAM1-exo and sictrl-exo.Co-culture with hIC AM 1-exo significantly promote the cell growth,proliferation,migration and invasion abilities of sictrl cells,and the ability of secreting IL-6,IL-8 and TGF-β1 in sictrl cells.The expression of ICAM1,Integrin β1 and MMP9 proteins of sictrl cells were up-regulated when treated by hICAM1-exo.The expression of RelB in the cytoplasm and nucleus of sictrl cells were slightly up-regulated after co-culture with hICAM1-exo.siRelB-exo had no significant effects on the biological behaviors,the expression of NF-κB signaling pathway and metastasis-related proteins in sictrl cells.Conclusions1.RelB mediated the transformation of fibroblasts into CAFs,indicating of Desmin and FAP expression,by upregulating exosomal ICAM1.2.RelB enhanced the malignant characteristics of tumor cells by upregulating exosomal ICAM1.Uptaking of exosomal ICAM1 may partly rescue the effects of RelB-knockdown on the biological behaviors of PC cells.3.RelB modified the PC microenvironment by upregulating exosomal ICAM1.This exosome-mediated cell-cell communication may be a molecular mechanism to promote PC metastasis. |
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