1、Accepted ManuscriptThe emerging role of exosome-derived non-coding RNAs in cancer biologyQing Fan, Shibo Wei, Liang Yang, Xiaodong Zhang, Xueqiang Peng, Dongming Su, Zhenhua Zhai, Xiangdong Hua, Hangyu LiPII:S0304-3835(17)30677-8DOI:10.1016/j.canlet.2017.10.040Reference:CAN 13583 To appear in:Cancer

2、 LettersReceived Date: 13 July 2017Revised Date:24 October 2017Accepted Date: 24 October 2017Please cite this article as: Q. Fan, S. Wei, L. Yang, X. Zhang, X. Peng, D. Su, Z. Zhai, X. Hua, H. Li, The emerging role of exosome-derived non-coding RNAs in cancer biology, Cancer Letters (2017), doi: 10.

3、1016/j.canlet.2017.10.040.This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is pub

4、lished in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPTTheemergingroleofexosome-derivednon-coding RNAs in cancer biologyQing Fan1, Shibo Wei1, Lia

5、ng Yang1, Xiaodong Zhang1, Xueqiang Peng1, Dongming Su2, Zhenhua Zhai3, Xiangdong Hua4, Hangyu Li1*1Department of General Surgery, The Fourth Affiliated Hospital of China MedicalUniversity, Shenyang, China2Center of Cellular therapy, the Second Afliated Hospital of Nanjing Medical University, Nanjin

6、g, China3DepartmentofOncology,TumourAngiogenesisandMicroenvironmentLaboratory (TAML), First Affiliated Hospital, Liaoning Medical College, Jinzhou, China4 Department of Hepatobiliary Surgery, Liaoning Cancer Hospital and Institute, Shenyang, ChinaAbstractExosomes are a new means of intercellular inf

7、ormation exchange that have aroused great research interest. Long neglected in research, exosomes were deemed nonfunctional cellular components to be discarded. However, it has been gradually revealed that exosomes are an important tool for the exchange of intercellular information and material. Exo

8、somes contain specific repertoires of non-coding RNAs(ncRNAs, including microRNA and lncRNA), indicating that a specific RNA sorting mechanism may exist. Correspondingly, intracellular multivesicular bodies (MVBs) are produced after fusion with the cell membrane to release exosomes rather than induc

9、ing autophagy, which reveals that there may be a specific regulatory mechanism for MVB secretion. Cells can trigger cancer-related disorders after the recognition and uptake of circulating exosomal ncRNAs, providing indications for early tumor biopsy and treatment. The use of exosomes as a biologica

10、l carrier in targeted therapy has been demonstrated. However, there may be a specific, unknown switch for loading drugs. This review focuses on the mechanisms of exosome biogenesis, release, and uptake. We also review the promotion of tumor development by exosomal ncRNAs including chemotherapy resis

11、tance，metastasis and the prospective use ofexosomes in cancer diagnosis and treatment.Keywords: Exosomes; MVBs; Tumor; miRNA; lncRNAAbbreviations:ncRNAs, non-coding RNAsTD-exosomes, tumor-derived exosomes TME, tumor and microenvironment DCs, dendritic cellsMHC, major histocompatibility complex hnRNP

12、A2B1, heterogeneous ribonucleoprotein A2B1SYNCRIP, synaptotagmin-binding cytoplasmic RNA-interacting protein MVBs, multivesicular bodiesSNARE, soluble N-ethylmaleimidesensitive factor attachment protein receptor SM, Sec1/Munc18Rab11FIPs, Rab11 family of interacting proteins ctDNA, circulating cell-f

13、ree tumor DNACTCs, circulating tumor cellsOSCC, oral squamous cell carcinoma1. IntroductionIn recent years, the tumor and microenvironment (TME) exchange of information in particular significantly affects tumor occurrence and development, as well as invasion, metastasis, and other malignant biologic

14、al behavior 1. Mainly, malignant phenotypic changes in solid tumors occur not only through direct contact and the secretion of soluble factors, but also through the secretion of exosomes, effecting the phenotype of malignant changes via microenvironment nourishment 2, 3. Remarkably, exosomes, a new

15、discovery in the intercellular communication medium, reveal important cellcell communication, and tumor cells secrete many exosomes to exchange information between local and distant cells.Exosomes are lipid vesicles that are 100 times smaller than cells and contain nucleic acids (genes, non-coding R

16、NAs ncRNAs, DNA), proteins, and lipids. Almost all cells secrete exosomes, which range 30100 nm in diameter 4-6. In 1981,Trams et al. found that shedding vesicles with an average diameter of 40 nm and ranging 5001000 nm in diameter could be isolated from various normal and tumor cells; this was the

17、first description of exosomes 7. In 1983, Pan and Johnstone and Harding et al. discovered and defined exosomes 6, 8. Exosomes were long considered an important metabolic pathway for cellular efflux, until it was shown that dendritic cells (DCS) play an important regulatory role by secreting exosomes

18、 containing major histocompatibility complex (MHC) and T cell costimulatory molecules 9. In 2007, Valadi found that exosomes can carry mRNAs and microRNAs (miRNAs) to transfer genetic material among cells to exchange information between close and distant cells 10. This discovery sparked research int

19、erest in the field of exosomes.The intensified study of exosomes has revealed that they act as bridges for important information exchange between cells, carrying nucleic acids, proteins, and lipids to the target recipient cells 11, 12. Tumor cells can treat exosomes as “garbage”, releasing them to p

20、romote tumor progression 13, 14. Overall, tumor cells can collectively release exosomes to achieve this aim. Here, we briefly outline some of the current knowledge on the mechanisms of exosome biogenesis, release, and uptake. We also highlight the critical effects exerted by exosomal ncRNAs on tumor

21、 progression and drug resistance, and the prospect of using exosomes in tumor diagnosis and treatment.2. ncRNA loading into exosomesThe transfer of ncRNA-loaded exosomes plays a key role in cellcell communication in many cancers 15, 16. Given the crucial function of Argonaute (AGO) proteins in ncRNA

22、s, they are considered fundamental ncRNA carriers and may be involved in ncRNA loading into exosomes. However, Gibbings et al. found that purified exosomes only contained single-stranded, mature miRNAs, high levels of GW182 (trinucleotide repeatcontaining 6A), and low levels of AGO2 protein, and det

23、ected neither P-body components nor miRNA-repressible mRNA in the exosomes 17. This suggests that miRNA loading into exosomes takes place in a miRISC (miRNA-induced silencing complex)-independent manner18. Consistent with this, Ostenfeld et al. did not observe any miRNA-processing proteins or miRISC

24、- associated proteins in exosomes 14. In their study, AGO2 was only detected in one of three replicates. Moreover, immunoblotting revealed no detectable AGO2. In addition, most AGO2 miRNAs were independent of exosomes 19, 20. These findings indicate that there may be other regulatory mechanisms for

25、ncRNAT IPRSCU A Nloadingintoexosomes.Fig.1. The mechanism of communication between cells by exosomes. ncRNAs contained different RNA motifs can be loaded into multivesicular bodies (MVBs) by different RNA-binding proteins. MVBs can either follow a degradation pathway fusingMwith lysosomes or release

26、 the ILVs as exosomes to the extracellular space. Recipient cells can uptake exosomes by three pathways: exosomal fusion,endocytosis and juxtacrine signaling.Recent studies have shown that some ncRNAs are enriched in exosomes while others are barely present, suggesting a potential regulatory mechani

27、sm for the sorting of specific sets of miRNAs into exosomes（Fig.1）. In T cells, miRNAs enriched in exosomes share the same specific sequence (GGAG), which has been identified as an EXOmotif. EXOmotifs are specifically recognized by hnRNPA2B1 (heterogeneous ribonucleoprotein A2B1) and hnRNPA1, thereb

28、y controlling the selective loading of such miRNAs into exosomes. Interestingly, hnRNPA2B1 is mostly sumoylated in exosomes, and sumoylated hnRNPA2B1 is essential for the sorting of miRNAs into exosomes 21. In hepatocytes, another common extra-seed sequence (GGCU, ahEXO motif) of miRNAs enriched in

29、exosomes mediates direct binding to the RNA- binding protein SYNCRIP (synaptotagmin-binding cytoplasmic RNA-interacting protein; also known as hnRNP Q or NSAP1) and controls the sorting of such miRNAs into exosomes 22. SYNCRIP knockdown impairs the exosomal loading of specific exosome-enriched miRNA

30、s. Different from EXOmotifs, hEXO motifs play a positive role in regulating miRNA localization. Embedding a hEXO motif into a miRNA, which is poorly present in exosomes, can enhance its loading into exosomes. Furthermore, although both hnRNPA2B1 and SYNCRIP can interact with a common exosome-sorting

31、 motif, they display sequence-specific exosomal sorting capacity in the loading of selected miRNAs 22. YBX1 (Y-boxbinding protein 1) is another protein that might bind specific RNA structural motifs, i.e., ACCAGCCU, CAGUGAGC, and UAAUCCCA of mRNAs, and long ncRNAs (lncRNAs) enriched in exosomes, and

32、 control specific ncRNA sorting into exosomes16 , shown in Fig.1.Therefore, ncRNAs are selectively loaded into exosomes. Specific proteins act in coordination with specific ncRNA sequences to control ncRNA sorting into exosomes. However, the regulatory mechanisms of ncRNA sorting into exosomes are u

33、nknown, so further research is warranted to determine the involvement of other RNA-binding proteins and RNA motifs.3. Exosome releaseThe intracellular generation of multivesicular bodies (MVBs) containing intraluminal vesicles (ILVs) is both ESCRT (endosomal sorting complex required for transport)-d

34、ependent and ESCRT-independent 15, 23, 24. After intracellular generation, how MVBs bind to a specific cell membrane region and through plasma membrane fusion ultimately achieve exosome release may involve a series of specific mechanisms of secretion. The mechanisms involved in secretory MVBs have b

35、een extensively studied.Exosome release involves several crucial factors and the synergistic effect of these factors is that MVBs bind to the cell membrane and is a key factor in achieving exosome release after plasma membrane fusion as shown in Fig.2. The most important factor is the small GTPases,

36、 including the Rab and RAL GTPases 25-27. The relatively in-depth study of the Rab GTPase family has found more than 70 subtypes located on different membranes of the surface. Rab GTPases respectively play related roles in vesicle budding, uncoating, motility, tethering, and fusion to coordinate the

37、 regulation of vesicle traffic 27.In general, the role of Rab is indispensable to the regulator and effector proteins 28, 29. Effector proteins, such as Sl272 and Slb2-b, are also required in Rab27- mediated vesicle transport and fusion 29, 30. The mechanism of Rab localization in various vesicles a

38、fter intracellular synthesis is uncertain 28, 31, but the localization of Rab in the vesicles and its recruitment of differently interacting effector proteins confers different biological functions on the vesicles 27. For example, the transition between early and late endosomes can be achieved byswi

39、tching SAND-1, or Mon1, in the Rab conversion process, and the loss of Rab5 is accompanied by the formation of inclusion bodies, marking the obtainment of late endosomal Rab7 32.So far, nine small GTPases are associated with secretion (Rab2B, Rab5, Rab7, Rab9A, Rab11, Rab27A, Rab27B, Rab35, RAL). Th

40、e Rab effect is inseparable from regulators and effectors, as shown by Rab35 and Rab27 29, 33. TBC1D10A-C (TBC1 domain family member AC) regulate the secretion of PLP-EGFP (proteolipid protein 1enhanced green fluorescent protein)-related exosomes by screening the Rab GAP library in exosome-secreting

41、 oligodendrocytes 33, and Rab27 binds to the corresponding effector proteins (Slp4-a, Slac2-b, Munc13-4) to regulate secretory vesicle transport and fusion 29.Rab mediates MVB transport and plasma membrane fusion by two categories of Rab effector proteins, respectively 29, 34. Obviously, in mediatin

42、g exosome secretion first, Rab is in the subcellular position of MVB directional transport, followed by the MVB and plasma membrane docking fusion 35. Within the cell, the actin and microtubule cytoskeleton is not randomly distributed, and exhibits significant polarity distribution 36, 37. Actin and

43、 Rab in granule secretion have been studied extensively 36, 38. Correspondingly, the targeted transport of MVBs is associated with the actin and microtubule cytoskeleton, being directly or indirectly bound to the actin and microtubule cytoskeleton, achieving polarized delivery of MVBs via mediation

44、by Rab and the corresponding effector on the MVB membrane and with the aid of a kinetic protein 29, 35 (Fig.2). Interestingly, Rab11and the Rab11 family of interacting proteins (Rab11FIPs) assist in vesicle transport with actin and kinetic proteins 39, which is defined as juxtanuclear recycling endo

45、somes. Moreover, it has long been confirmed that Rab27A interacts with the corresponding effector (Slac2-a) and the actin-based motor myosin to transport melanosomes along actin filaments 29. In addition, invadopodia, actin-rich subcellular structures formed by invasive cancer cells that protrude an

46、d degrade extracellular matrix (ECM), are specific for Rab27a, CD63-positive MVBs, and key docking and secretion sites; cortactin has a stabilizing effect on invadopodia, and can further enhance the MVB docking site to promote exosome secretion. On the contrary, inhibiting cell-formation invadopodia

47、 significantly reduced exosome secretion, all of which indicates the pivotal role of actin in exosome secretion 40, 41.MVBs not only require the kinetic action of motor proteins in plasma membrane directional transport, but also cannot be separated from the microtubule cytoskeleton “railroad”, MVBs

48、move along the microtubule network to the microtubule plus ends to ultimately achieve MVB and plasma membrane docking fusion 36, 42. Nevertheless, the specific mechanism spanning MVB and plasma membrane docking fusion to the final release of exosomes is not clear. However, it is currently believed t

49、o involve the following steps shown in Fig.2.T IPRSCUFig.2.Transport and membrane fusion of the secretory MVBs. Rab GTPases can mediate MVBs transport along actin cytoskeleton (cytoskeletal tracts). When the secretory MVBs close to the plasma membrane, Rab GTPases can promote MVBsadhering by recruit

50、ing tethering factors in the target membrane. Meanwhile, the SM protein-bound t-SNAREs is assembled with v-SNAREs to activate membrane fusion.Small GTPases and the exocyst complex mediate MVB membrane docking to directly or indirectly initiate SNARE complex assembly 43 as shown in Fig.2. Rab27 and t

51、he corresponding effector complex play a key role in MVB plasma membrane docking 29. In addition, Rab27A knockout cells have excessive cortactin expression and unaltered exosome secretion; therefore, Rab27A may be associated with cortactin in mediating MVB docking 29, 41. At the same time, the exocy

52、st complex is critical for the assembly and control of SNARE complexes 43. Sec3(exocyst complex component 1, belonging to the exocyst complex) interacts directly with the target membrane SNARE (t-SNARE, SYX-5) protein Sso2 to initiate t- SNARE assembly plasma membrane fusion 44. However, RAL1-mediat

53、ed MVB membrane mating and fusion are often independent of the exocyst complex, and theactive form of RAL1 can activate or recruit SYX-5 at the top plasma membrane to promote MVB fusion, thereby promoting exosome release. Furthermore, when SYX- 5 is absent, MVBs accumulate under the plasma membrane

54、25.Vesicular SNARE (v-SNARE) and t-SNARE proteins catalyze the fusion of the two membranes (Fig.2), and the ATPase NSF (N-ethylmaleimide sensitive factor) and its adapter proteins disassemble the SNARE complex to recycle SNARE for another round of fusion 45-47. In K562 cells, VAMP7 (vesicle-associat

55、ed membrane protein 7, a v-SNARE protein) is involved in MVB plasma membrane fusion and exosome release 47. In mammals and nematodes, SYX-5 is also involved in MVB fusion to promote exosome release 25. Tumor cells are usually aerobic glycolytic, and the key enzyme PKM2 (muscle pyruvate kinase) can f

56、urther stabilize the SNARE complex through SNAP-23 (synaptosome-associated protein 23, t-SNARE) Ser95 phosphorylation to promote exosome release 48. More interestingly, invadopodia are a key exosome docking site 40. Additionally, invadopodia formation and maintenance requires the pairing of VAMP7 an

57、d SNAP23, and syntaxin 4 (t-SNARE) mediates MMP (matrix metalloproteinase) trafficking to the invadopodia 49. In summary, these findings show that SNARE is at least partially involved in invadopodia formation, which indirectly affects exosome secretion and suggests that SNARE is involved in plasma m

58、embrane docking fusion 40, 49. The ATPase NSF and its adapter protein, which dismantle the SNARE complex to recycle SNARE, are also necessary for plasma membrane fusion 45, 47, 50.As shown in Fig.2, SM protein is a soluble factor that may interact with SNARE before and after vesicle attachment. The hook-shaped SM binds to the cell membrane durin