| BackgroundRenal cell carcinoma(RCC)and prostate cancer(PCa)are the common cancer in the genitourinary.Our paper focus on the RCC and PCa.Renal cell carcinoma(RCC)is a form of renal epithelial cancer that makes up 85%of primary renal tumors and 3%of all malignant tumors in adults.It remains the fifth most frequently detected cancer type,and accounts for approximately 2.5%of cancer-associated mortality in Europe.Globally,there were 403,262 new RCC Diagnoses and 175,098 deaths,with the mortality rates associated with this tumor having risen to high levels that have stabilized in the past decade.Many prior studies have explored genomic and epigenetic mechanisms responsible for controlling the development and progression of RCC and the surrounding immune response.In contrast,fewer studies have explored the microenvironment of RCC-associated tumor microenvironment,and the specific regulatory roles of extracellular vesicles(EVs)remain poorly understood.EVs are small multivesicular bodies(30-150 nm in size)that are released from almost all mammalian cells.In general,higher EV production has been observed in cancer cells relative to healthy control cells,with these particles playing important roles in promoting or inhibiting oncogenesis,metastasis,and immunity.These EVs can encapsulate different macromolecules and signaling intermediates including nucleic acids(mRNA,microRNAs[miRNAs],and DNA),proteins,lipids,and metabolites,allowing for the easy trafficking of these molecules between cells.As miRNAs within EVs are stabilized by the surrounding lipid bilayer,they are of particular interest in the context of the oncogenic importance of these vesicles.Owing to their rapid growth in size,many tumors exhibit varying degrees of hypoxia that are closely linked to tumor progression.RCC tumors often exhibit such hypoxic regions,and they have been linkedto both therapeutic resistance and poorer patient survival.As such,hypoxia represents a valuable model for studies of the impact of the tumor microenvironment on RCC progression,allowing for further analysis of the roles of miRNA in EVs.The present study was therefore designed with the goals of isolating purified RCC-associated EVs,determining whether EVencapsulated miR-155 plays a role in hypoxia-mediated induction of RCC progression,and examining the mechanisms whereby miR-155 impacts RCC.Prostate cancer is identifed as a type of the most common male malignancies in the world,with an increasing incidence and mortality in recent years.The epidemiological survey shows that in the past 10 years,the developed degree of a country is negatively correlated with the death rate of PCa patients,that is,the more backward the country,the higher the fatality rate of PCa.Considering the clinical value of PCa,the occurrence of tumors and efective treatment methods need to be studied in-depth.Long non-coding RNAs(ncRNAs)were initially identifed as the "garbage" of genomic transcription.Nevertheless,recent researches have elucidated that IncRNAs are involved in regulating molecular processes,such as X-chromosome silencing,gene imprinting,chromatin modifcation,transcriptional activation,transcriptional interference,and intra-nuclear transport,which begin to attract widespread attention[5-10].During the development of PCa,lncRNAs play an important regulatory role.For instance,androgen-induced IncRNA SOCS2-AS1 facilitates PCa cell proliferation and prohibits apoptosis.LncRNA MALAT-1 is recognized as a newly-found possible therapy target for PCa with castration resistance.Low BDNF-AS expression is related to the unsatisfactory prognosis of PCa patients.Further,LINC00689 has recently drawn attention when studying its role in cancer progression.However,the number of the concerned research is limited.Terefore,the regulation mechanism of LINC00689 in PCa remains a novel topic of concern in this study.In our research,LINC00689 promotes cell proliferation,migration,invasion as well as suppresses cell apoptosis via regulating miR-496/CTNNB1 to activate Wnt pathway,which may contribute to fnd a fresh target for PCa treatment.Objectives1.Evaluate the impact of EVs from RCC patients on renal cell carcinoma cells.2.To explore whether hypoxia can promote the production of EVs by renal carcinoma cells.3.To explore whether Hypoxia induced EVs can promote renal carcinoma progression.4.Find out the sigal pathways that miR155 in EVs promotes the progression of RCC through.5.To assess the expression of LNC00689 in PCa and its impact on PCa progression.6.To explore the impact of LNC00689 knockdown on PCa progression.7.To find the miRNA and the downstream target regulated by LINC00689.8.Find out the sigal pathways that LNC00689 promotes the progression of PCa through.Methods1.In total,we enrolled 132 RCC patients that had been newly diagnosed at Qilu Hospital between December 2017 and August 2018.miR-155 exprossion levels were evaluated by qt-PCR.Furthermore,we assessed the impact of EVs from RCC patients on 786-0 and Caki-1.2.We assessed the morphology of these EVs via electron microscopy,and we analyzed their expression of surface marker proteins including TSG101(Tumor susceptibility protein 101)and CD9(Cluster of differentiation 9)via Western blotting Additionally,we measured the size and uniformity of these EVs in a nanoparticle tracking analysis.3.We assessed protein levels within EVs derived from Caki-1 and 786-0 cells after hypoxia treatment.miR-155 levels both in cells and EVs were evaluated by qt-PCR.To further confirm that this miR-155 was encapsulated in EVs derived from these RCC cells,we treated cellular supernatants with either RNase A and/or Triton X-100.4.We collected EVs from RCC cells grown under hypoxic conditions as above,labeled them with the red fluorescent PKH26 dye,and then added them to 786-O and Caki-1 cells for 72 h.We then used a CCK-8 assay to assess the viability of these cells.The migratory capabilities of these cells were then assessed in a wound healing assay.The impact of these EVs on cell cycle progression and apoptosis was also assessed by flow cytometry.5.We transfected cells with miR-155 mimics or inhibitors prior to culturing them under hypoxic conditions and assessed the viability of these cells.To explore the mechanistic basis.6.Whereby miR-155 modulates RCC cell proliferation,we conducted a predictive bioinformatics analysis to identify potential miR-155 target genes.Based on this target sequence,we then constructed luciferase reporter constructs containing either a WT or mutant version of this binding site and then used them in a luciferase reporter assay.At the end,we assessed the functional impact of altered FOX03 expression on RCC progression after hypoxia-and normoxia-induced EV treatment.7.80 patients chosen from Qilu Hospital of Shandong University were included in this research.Following surgical resection,tumor tissues were quickly frozen in liquid nitrogen and subsequently stored at-80℃ for further use.8.Normal prostate epithelial cell(RWPE1)and PCa cells(DU145,LNCaP,PC-3 and C42B)were cultured in line with previous description.They were cultured with 10%FB S and 1%antibiotics in DMEM.In order to activate the Wnt/β-catenin signaling pathway,DU145 cells were treated with lithium chloride for 24 h.9.Plasmids were constructed:Specific shRNAs against LINC00689,as well as the pcDNA3.1 vector containing the whole sequence of LINC00689 or CTNNB1 and the empty vector,the miR-496 mimics,miR-496 inhibitors,NC mimics and NC inhibitors.By use of Lipofectamine 3000,plasmids mentioned were individually transfected into DU 145 or LNCaP cells in 24-well plates for 48 h.10.Total RNAs were extracted from tissues or cells by utilizing TRIzol reagents,and then reverse-transcribed into cDNA in line with the protocol of a reverse transcriptase kit.Next,RT-qPCR was undertaken with PCR system.Relative gene expression was normalized to GAPDH or U6.Quantification of relative gene expression was conducted via comparative 2-ΔΔCt approach.11.Cell viability was explored with the MTT assay.Cell proliferation was explored with colony formation assay.The Annexin V-FITC Apoptosis Detection Kit was employed to examine cell apoptosis rates.Transwell assay was used to assess the invasion or migration ability.12.Acorrding to the prediction of starBase v3.0,LINC00689 or CTNNB1 3’UTR fragments covering wild-type and mutant miR-496 binding sites were inserted into the pmirGLO dual-luciferase plasmid.Dual luciferase reporter assay system was applied to detect luciferase activities.13.The RIP assay was implemented via a Magna RIP RNA Binding Protein Immunoprecipitation Kit.Detection of the enriched RNA was subjected to RT-qPCR.14.Transfected DU145 or LNCaP cells protein expressions were asessed by WB with primary antibodies against CTNNB1,β-catenin,CCND1,CDK2,c-MYC,and GAPDH.15.All assays were undertaken in triplicate.P<0.05 was considered statistically significant.For analyzing the experimental data which were expressed as means ±SD,SPSS 17.0 software was used.Student’s t-test was conducted for comparing two groups,and one-way ANOVA was for comparing multiple groups.Survival rate was assayed with Kaplan-Meier approach,and difference was analyzed by a log-rank test.Results1.Hypoxia-induced microRNA-155 overexpression in extracellular vesicles promotes renal cell carcinoma progression by targeting FOXO3miR-155 expression levels in EVs isolated from RCC healthy control patients,are at significantly higher levels compared to those healthy control patients.In addition,we found that the expression level of this miRNA rose with RCC progression.Cell viability was enhanced in the presence of EVs from RCC patients.EVs from RCC patients markedly augmented the wound closure relative to EVs from healthy control patients when used to treat RCC cell lines.Furthermore,EVs from RCC patients alleviated 786-O and Caki-1 cell apoptosis.miR-155 levels were also elevated in RCC cell lines after treatment with EVs from RCC patients.Hypoxia significantly increased protein levels in EVs and miR155 level both in cells and EVs.Hypoxia induced RCC cell-derived EVs labeled with the red fluorescent PKH26 dye were taken into the cells.viability and migratory capabilities were significantly bolstered by those EVs.The impact of these EVs on cell cycle progression and apoptosis show that it enhanced cell proliferation and alleviated 786-O and Caki-1 cell apoptosis.In addition,we found that miR-155 expression levels in 786-O and Caki-1 cells were 74.8%and 36.9%higher,respectively,when these cells were treated with hypoxia-derived EVs relative to when they were treated with normoxia-derived EVs.We confirmed that miR-155 mimics and inhibitors were able to significantly alter miR-155 expression in the expected directions in both 786-O and Caki-1 cells.EVs derived from miR-155 mimic-transfected cells were able to more effectively enhance cell viability,whereas the opposite was observed when cells were treated with EVs derived from miR-155 inhibitor-transfected cells.miR-155 mimics were able to significantly reduce WT but not mutant FOXO3 3’-UTR reporter activity in both 786-O and Caki-1 cells.miR-155 mimic transfection was associated with a significant reduction in FOXO3 expression,whereas the opposite was observed in response to miR-155 inhibitor transfection.Hypoxia-induced EVs were able to significantly reduce nuclear and cytoplasmic FOXO3 expression.Furthermore,hypoxia-induced EVs can also decrease the phosphorylation levels of FOXO3 to inhibit its activation.FOXO3 target genes,including both proliferation related genes(p15,p21,and Gadd45)and apoptosis-related genes(Bim and TRAIL)were further downregulated by hypoxia-induced EV treatment.2.LINC00689 promotes prostate cancer progression via regulating miR-496/CTNNB1 to activate Wnt pathwayThe expression of LINC00689 was much higher in PCa tissues than that in adjacent normal tissues.Meanwhile,LINC00689 expressed much higher in PCa cells than control cells.The patients with PCa at the advanced stage(Ⅲ-Ⅳ)possessed much more LINC00689 expression than the patients at an early stage(Ⅰ-Ⅱ).High LINC00689 expression was closely associated with short overall survival time of PCa patients.MTT assay,colony formation assay and transwell assay validated that silencing of LINC00689 suppressed cell proliferation,migration and invasion in DU 145 and LNCaP cells.Flow cytometry detected that LINC00689 downregulation induced about two folds of increase in cell apoptosis rate in DU 145 and LNCaP cells.Upregulated protein level of cleaved caspase 3 was caused by LINC00689 knockdown.Acorrding to starBase v3.0,miR-496 was predicted as the miRNA sponged by LNC00689.The binding relationthe was confirmed by Luciferase reporter assay.The rescuing function of miR-496 inhibitor in sh-LINC00689 transfected cells was elucidated by MTT,colony formation assay,apoptosis assay,western blot analysis and transwell assay.The expression of the indicated nRNAs in normal prostate epithelial cell(RWPE1)and PCa cells(DU 145 and LNCaP)was detected by RT-qPCR.CTNNB1 was prominently upregulated in DU145 and LNCaP cells.In hence,CTNNB1 was chose to perform the following assays.RIP assay and luciferase reporter assay verified the binding facts between miR-496 and CTNNB1.pcDNA3.1/CTNNB1 was conducted and transfected into DU145.TOP/FOP fash assay suggested that pcDNA3.1/CTNNB1 or LiCl treatment offset the interceptive role of sh-LINC00689 in Wnt pathway.RT-qPCR and western blot assays detected that mRNA and protein level of Wnt-related proteins were reduced by knocking down LINC00689,then counteracted by CTNNB1 upregulation.Through MTT and colony formation assays,upregulation of CTNNB1 reversed the inhibiting effects of knocking down LINC00689 on cell proliferation.In a similar way,LINC00689 silencing spurred DU145 cell apoptosis,and this stimulating effect was partly offset by overexpressing CTNNB1.Transwell assay proved the rescuing role of pcDNA3.1/CTNNB1 in DU145 cell migration and invasion restrained by LINC00689 insufficiency.Conclusions1.miR-155 in EVs from RCC patients can promote RCC progression.2.Hypoxia was sufficient to enhance EV secretion and promote increased miR-155 expression in RCC cells and in EVs derived therefrom.3.Hypoxia can drive RCC progression through a mechanism that is associated with miR-155 upregulation.4.miR-155 is able to directly suppress FOXO3 expression in RCC cells and promote the progression of RCC.5.LINC00689 is upregulated in prostate cancer tissues and cells6.Knock down of LINC00689 restrains prostate cancer progression7.LINC00689 sponges with miR-496 in prostate cancer and miR-496 targets to CTNNB1 in prostate cancer.8.LINC00689 activates Wnt pathway via upregulating CTNNB1.9.LINC00689 promotes prostate cancer progression by upregulating CTNNB1... |
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