| Background:Prostate cancer is a common malignancy with high mortality.In 2017,an estimated 164,690 new cases of prostate cancer were diagnosed in the United States and 29,430 people will die from this disease.The incidence of prostate cancer depends on geographical location and ethnical background.In China,the risk of prostate cancer is lower than that in western countries.However,the risk has been increasing rapidly with a growing and aging population in China over the past few years.For cancer diagnosis and treatment,biomarkers begin to be used in the past half century.The new diagnostic biomarkers such as prostate specific antigen(PSA)have substantial drawbacks such as false-negatives,falsepositives and lack of tumor-type specificity.Tumor biopsy is the only definitive method of diagnosis,but it is invasive.Novel prostate cancer biomarkers are required for clinical application.Accumulating evidence has showed that Nanosized vesicles-exosomes mediate the interaction between cancer cells and their microenvironment and it is critical in the development of cancers.Exosome can be secreted from multiple types of cells and can be extracted from human body fluids such as urine,plasma and saliva.Exosomes retain proteins,lipids,DNA,m RNAs,micro RNAs and lnc RNA,which can be transported to nearby or distant normal or abnormal cells.Then,the local and long-range transportation may influence on the key signaling pathways in cancer.Long noncoding RNAs(Lnc RNAs)are endogenous transcribed RNA molecules that lack an open reading frame encoding a protein.Lnc RNAs are ranging from 200 to 100,000 nucleotides and distinct from mirco RNAs.Lnc RNA can regulate gene expression through recruiting chromatin-modifying complexes and interacting with mi RNA,m RNA or protein.Lnc RNA modulate six characteristics of cancer,including proliferation,growth suppression,motility,immortalization,angiogenesis and other cancer phenotypes.Therefore,previous reports suggest that lnc RNA in exosomes can promote tumorigenesis.Cells can be trigger cancerrelated disorders after the recognition and uptake of circulating exosomal lnc RNAs,providing indications for early tumor biopsy and treatment.Thus,the identification of key lnc RNAs derived from exosome will benefit the practical research of prostate cancer mechanism.In our study,we sought to identify the functional role of exosomal lnc RNA involved in prostate cancer progression.We identified key lnc RNA expression by lnc RNA array of exosomes which derived from different prostate cell line supernatant.We found that one lnc RNA in particular,DANCR,showed excessive aberrant expression in prostate cancer cell lines exosome.Several research groups have demonstrated that DANCR may serve as biomarkers in various developmental and biologic processes,as well as in cancer progression.The goal of the current study was to characterize the function of exosomal DANCR and its ability to mediate bone microenvironment.Methods: 1.To isolate exosome from the supernatant of normal cells BPH-1,RWPE-1 and prostate cancer cells C4-2B,VCa P,and PC3 by ultracentrifugation;2.To isolate exosome from clinical collected plasma samples whichincluding 7 prostate cancer patients and 7 non-tumor normal plasma by ultracentrifugation;3.To identify exosomes by Nanosight and Western blot;4.To analyze lnc RNAs differentially expression in exosomes which derived from prostate cancer cell lines by Lnc Finder RT2 lnc RNA PCR Array;5.To identify DANCR expression in exosomes derived from different prostate cancer cells by q RT-PCR;6.To determine DANCR expression in prostate cells and serum exosome by q RT-PCR;7.To determine whether ST2 cells treated with exosomes affect PC3 cells proliferation by WST-1 assay;8.To measure DANCR expression in ST2 cells treated with exosomes derived from epithelial cells or cancer cells by q-PCR;9.To construct stable DANCR knockdown prostate cancer cell lines PC3,C4-2B and VCa P by lentiviral sh RNA;10.To measure DANCR expression in ST2 cells treated with DANCR knockdown exosome by q-PCR;11.To explore condition medium from ST2 cells treated by exosomes with DANCR knockdown affect prostate cancer cellsproliferation by WST-1 assay;12.To explore the biologic function of DANCR in regulating tumor microenvironment by transwell assay;13.To utilize western blot and immunofluorescence microscopy analysis to examine the autophagy in ST2 treated with exosome which with or without DANCR;14.To screen potential Cytokines or Chemokines in ST2 treated with exosome by Mouse Cytokines & Chemokines Array.Results: A.Screening specific lnc RNA of prostate cancer cell derived exosomes.1.The most prevalent population of particles ranged from 50-150 nm in diameter and average size around 100 nm were identified.The identity of the exosome was confirmed by detecting three typical exosome specific marker proteins— Alix,TSG101 and HSC70.We validated the method of exosome isolation from prostate cancer cell lines supernatants and plasma samples.2.Compared with BPH1-Exo,we identified some lnc RNAs dysregulated in C4-2B-Exo or VCa P-Exo.There are DANCR,SNHG16,IPW,UCA1 and RMST.There are five lnc RNAs dysregulation in C4-2B-Exo or VCa P-Exo compared with the control RWPE-1 Exo,including DANCR,SNHG16 and IPW,TUG1,and UCA1.3.After q RT-PCR validation,we found that DANCR expression in exosome derived from prostate cancer cell line was consistent with the lnc RNA Array screening results.Exosomes derived from control cells have lower DANCR expression,but showing significant higher expression in prostate cancer cells.4.The expression DANCR in prostate cancer patient plasma exosomes is higher than that in non-cancer normal plasma exosomes.B.DANCR derived from prostate cancer cells exosomes can be incorporated into recipient ST2 cells and involved in tumor microenvironment.1.WST-1 results show that conditioned medium(CM)from the ST2 cells treated with prostate cancer cells exosomes promoted PC3 cells growth.2.DANCR expression was increased in ST2 cells after treated with exosomes from prostate cancer cells.3.All sh RNA constructs decreased DANCR expression by over 80% compared with that of scramble-non-target sh RNA as validate by q RT-PCR.4.The scramble-Exo treatment group had higher DANCR expression,compared with the control group or DANCR knockdown group.C.Exosome promotes prostate cancer cell perliferation,migration and invasion through DANCR in regulating BMSC.1.CM from ST2 cells treated by C4-2B exosomes with DANCR knockdown inhibited the proliferation of prostate cancer cells.2.CM from ST2 cells treated by PC3 exosomes with DANCR knockdown impaired cellular migration ability promoted by PC3 exosome in PC3 cells.3.CM from ST2 cells treated by C4-2B exosomes with DANCR knockdown impairs cellular migration ability promoted by C4-2B exosome in PC3 and VCa P cells.4.CM from ST2 cells treated by C4-2B exosomes with DANCR knockdown impairs cellular invasion ability promoted by C4-2B exosome in PC3 cells.D.DANCR regulates bone stromal cells by mediating autophagy.1.The expression of LC3-II and proportion of autophagic vacuoles cells were decreased in ST2 cells after treated with C4-2B exosomes without DANCR.2.CCL4 expression of was decreased in ST2 cells after treated with C4-2B exosomes without DANCR.Conclusion: We found that one lnc RNA in particular,DANCR,showed excessive aberrant expression in prostate cancer cell lines exosomes.The expression of DANCR was high not only in prostate cancer cell lines exosomes,but also in its parental cells.In addition,DANCR expression level in clinically collected prostate cancer patient plasma exosomes is higher than that in non-tumor healthy plasma exosomes.These results may suggest an increased expression of DANCR in prostate cancer.DANCR can be incorporated into recipient ST2 cells via exosomes and involved in bone microenvironment.Conditioned medium from ST2 cells treated with DANCR knockdown exosome led to decreased prostate cancer cell proliferation,migration and invasion in vitro.In summary,exosomes promote prostate cancer progression through DANCR-mediated regulation of the bone microenvironment. |
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