1、EUROPEAN UROLOGY 67 (201 5) 33-4 1Article history:Accepted July 28. 2014Keywords:Exosome microRNA Extracellular RNA RNA sequencing Prostate cancer Biomarker Prognosis SurvivalCrossMarkavailable at journal homepage: European Association of UrologyPlatinum

2、Priority - Prostate CancerEditorial by Benedikta S. Haflidaddttir and Yvonne Ceder on pp. 42-43 of this issueExosomal miR-1290 and miR-375 as Prognostic Markers in Castration-resistant Prostate CancerXiaoyi Huangabf Tiezheng Yuana9 Meihua Liangc9 Meijun Du a9 Shu Xia Rachel Dittmaraf Dian Wange9 Wil

3、liam See f9 Brian A. CostelloFernando Quevedos9 Winston Tan Debashis Nandys9 Graham H. Bevan Sherri Longenbach8, Zhifu Sun Yan Lu Tao Wang9 Stephen N. Thibodeau m9 Lisa Boardman8, Manish Kohli8 Liang Wanga td Department of Pathology and Cancer Center, Medical College of Wisconsin. Milwaukee. Wl. USA

4、;b Hiotherapy Center. Third Affiliated Hospital of Harbin Medical University, Harbin, China：c Department of Endocrinology, Second Affiliated Hospital of Harbin Medical University. Harbin, China： d Department of Oncology. Tongji Hospital ofTongji Medical College. Huazhong University of Science and Te

5、chnology, Wuhan. China：e Department of Radiation Oncology, Medical College of Wisconsin, Milukee. Wl. USA; department of Urology, Medical College of Wisconsin, Milwaukee, W/. USA;s Depart ment of Oncology. Mayo Clinic, Rochester. MN. USA： h Department of Oncology, Mayo Clinic, Jacksonville. FL, USA：

6、 University of Rochesrer Medical Center. Rochester. NY. USA;j Division of Biomedical Statistics and Infomiatics. Mayo Clinic. Rochester, MNt USA： k Department of Physiology. Medical College of Wiscons譏 Milwaukee. IV/. l/SA; division of Biostatistics Medical College of Wisconsin Milwaukee. WI. USA： n

7、, Department of Laboratory Medicine and Pathology Mayo Clinic. Rochester, MN. USAAbstractArticle infoBackground: Extracellular microRNAs (miRNAs) embedded in circulating exosomes may serves as prognostic biomarkers in cancer.Objective: To identify and evaluate plasma exosomal miRNAs for prog nosis i

8、n castrati on-resis tant prostate cancer (CRPC).Design, setring, and participants: RNA sequencing was performed co identify candidate exosomal miRNAs associated with overall survival in a screening cohort of 23 CRPC patients. Candidate miRNAs were further evaluated for prognosis using quantitative r

9、eal-time polymerase chain reaction in a follow-up cohort of 100 CRPC patients.Outcome measurements and statistical analysis: Cox regression and Kaplan-Meier survival analyses were used co evaluate survival association using candidate miRNAs along with clinical prognostic factorsResults and limitatio

10、ns: RNA sequencing in screening cohort generated approximately 6.80 million mappable reads per parien匸 Of those with nonnalized read counts 5.43% were mapped to miRNAs for a total of 375 known and 57 novel miRNAs. Cox regression analysis identified an association of miR-1290.-1246. and -375 with ove

11、rall survival (false discover rate 0.05). Of those, higher levels of miR-1290 and 375 were significantly associated with poor overall survival (p 0.004) in the follow-up cohort. Incorporation of miR-1290/-375 into putative clinical prognostic factors-based models in CRPC stage significantly improved

12、 predictive performance with a timedependent area under the curve increase from 0.66 to 0.73 (p 6.57 x 10 6).Conclusions: Plasma exosomal miR-1290 and miR-375 are promising prognosric biomarkers for CRK patients Prospective validation is needed for further evaluation of these candidate miRNAs. Patie

13、nt summary: In this study. we evaluated whether small RNAs circulating in blood could be used to predict clinical outcomes in late-stage prostate cancer patients. We identified two blood- based small RNAs whose levels showed significant association with survival. Our results warrant further investig

14、ation because the noninvasive bloodbased test has great potential in the management of late-stage prostate cancer.r .Mayo Clinic. 200 First Street. SW. Rochester. MN 55905. USA. Tel. +1 507 284 3903.E-mail addresses: (L. Wang), (M. Kohli).http:/dx.doi.Org/10.1016/j

15、.eururo.2014.07.0350302-2838/( 2014 European Association of Urology. Published by Elsevier B.V. All rights reserved.#EUROPEAN UROLOGY 67 (201 5) 33-4 11. IntroductionProstate cancer (PCa) is the second leading cause of cancer- rclatcd mortality in men, with 240 000 newly diagnosed cases and 29 000 d

16、eaths annually in the United States 1.In advanced metastatic stages, androgen deprivation therapy (ADT). a standard first-line therapy, leads to substantial cancer control and palliation that lasts for a nledian time of 20-30 mo |2V3. After failure of ADT and emergence of a castrationn?sistant stage

17、, circulating tumor cell counts (CTCs) are the only prognostic biomarker approved by the US Food and Drug Administration. This test is technically challenging and expensive with prognosis adversely affected for patients with 5 CTCs per 7.5 ml blood 4,5. With the in creased availability of therapeuti

18、c optio ns for this stage, access and clinical utility of easy-to-use prognostic and predictive biomarkers has become clinically relevant. Cell free nucleic acid profiling such as exosomal RNA in circulatory fluids offers an attractive potential for prognos tic and predictive biomarker development (

19、6|.Exosomcs arc small (30-100 nm) membrane vesicles released into the extracellular environment on fusion of multivesicular bodies w讓h the plasma membrane |7). It is reported that exosomes contain RNAs selectively enriched from parent cells 8, A number of plasma exosomal RNAs are reported as diagnos

20、tic, prognostic or even therapeutic biomarkers in cancer patients 6|. miR-107. miR-574-3pt miR-375 and miR-141 are upregulated in metastatic PCa patients |9J. Circulating microRNA (miRNA)-375 and miRNA-141 are correlated with a high Gleason score or lymph-node positive status 10,11, implying great p

21、otential as a PCa biomarker. We recently developed a comprehensive pipeline for exosomal RNA profiling analysis including exosome isolation. RNA extraction. library preparation, sequencing, and bioinformatics analyses 12,13. In this study, we applied this pipeline to examine exosomal RNA profiles ba

22、sed on the hypothesis that plasma exosomal RNAs can be used to predict survival in castration-resistant prostate cancer (CRPC) patients2. Material and methods2.1. Patients and samplesPatients consented to participate in an advaneed PCa biomarker registry prior to blood collection at the time of ADT

23、failure For plasma specimens whole blood collection was first centrifuged at 2000 rpm for 10 min. The supernatant was then fractioned into multiple aliquots for storage at -80 C. A screening cohort (n = 23) and a follow-up cohort (n = 100) were included. ADT failure was defined as the date of the fi

24、rst occurrence of either two serial prostate-specific antigen (PSA) rises during continuous ADT measured at least 1 wk apart with an absolute PSA level 2 ng/ml14) or the appearance of new image-based metastasis during ADT or initiation of castration-resistant specific therapy to ongoi ng ADT and in

25、the presence of subcastration testosterone levels (16 nt were aligned against miRBase (Release 19. 2043 entries) and human genome (Release 103). miRNA profiling was normalized using read counts per million mappablc miRNA sequences miRDccp2 15 was used co identify novel miRNAs from sequencing data. P

26、redicted iniRNAs with scores 2 were considered significan匸2.4. Quantitative reverse transcription polymerase chain reactionTo validate miRNAs identified by RNA sequencing. TaqMan MicroRNA Assays (Ufe Technologies) and miScript SYBR Green PCR Kit (Qiagen, Valencia, CA. USA) were used to measure expre

27、ssion levels of miR-30a/c 5p. miR-99a-5p. le(-7c. miR-1246. miR-1290. miR-16-5pv miR-125a-5p. and miR-375 in the plasma exosomal RNAs. Among tliose. miR-30a/e-5p. miR-99a-5p. miR-1 &5p. miR-125a-5p. and let-7c were evaluated as candidates for normalization control. PCR reaction was performed in tnpl

28、icate and analyzed using an ABI 7900HT system. Qiagen miScript 11 RT kit was also used for validation of miR-375. miR-30a/c-5p. and miR-16-5p.2.5. Identification of endogenous referencesIn the screening stage. the normalized sequencing data from 192 individuals (23 with CRPC and an additional 169 he

29、althy and nonhealthy subjects) were first screened for the most stably expressed miRNAs with an overall coefficient of variation (CV) 5%. Selected miRNAs were further processed by NomiFindcr 16 and Best Keeper 17, Diagnostic power with p 16 nt (query sequences) were approximately 10 million (76.60%)

30、. Of these, 6.7 million reads (51.42%) were mapped to known RNA species and the human genome. Among the mapped reads, mature miRNAs were the most common with an average of 2 784 360 reads (41.72%), followed by piwi-interacting RNA with 1 396 493 (20.92%), long noncoding RNA with 1 347 486 (20.19%),

31、and messenger RNA with 434 955 (6.52%). Six other RNA species collectively constituted 10.64% of the mappable sequences (Fig. 1; Supplementary Table 1). In this study, we reported our analysis on 375 comm on miRNAs and 57 putative miRNAs with 5 counts per million mappable reads (Supplementary Table

32、2). The raw sequencing data were deposited in the Gene Expression Omnibus database (accession number: GSE58410).3.2. Association of overall survival with exosomal microRNAsTo identify survival-related exosomal RNAs in CRPC patients, we further excluded those miRNAs w讓h log2Mappable Reads6 673 966 51

33、.42%piRNA20.92%Query Reads9 941 800 76.60%premature miRNA 2.74%Sq.93%q gg%NA f蠱pseudogene 3.10% tRNFig. 1 - RNA species and their distributions in plasma exosomes. Raw reads are the sequences detected by RNA sequencing. Query reads are those after trimming. Mappable reads are those mapped to known h

34、uman RNA or genome.IncRNA = long noncoding RNA; miRNA = micro RNA; piRNA = piwi-interacting RNA; rRNA = ribosomal RNA; snRNA = small nuclear RNA; snoRNA = small nucleolar RNA; tRNA = transfer RNA.mRNA6.52%IncRNA20.19%mature miRNA41.72%transformed read counts 5. We performed Cox regression analysis u

35、sing the remaining 248 known and 19 predicted miRNAs. We identified three miRNAs with a p value 0.0008 (false discovery rate 0.05) including miR-1290 (hazard ratio HR: 1.79; 95% confidence interval CI, 1.30-2.48). miR-1246 (HR: 8.04; 95% Cl, 2.36-27.33), and miR-375 (HR: 2.69; 95% CI, 1.52-4.77). In

36、 addition, we also observed an association of other RNA types with survival (Supplementary Table 3). For miRNAs, we dichotomized these patients using miRNA mean expression values as a cut-off and performed Kaplan-Meier analysis. We confirmed the significant association of miR-1290, miR-1246, and miR

37、-375 w讓h overall survival (p 0.05) (Supplementary Fig. 1).3.3. Candidate microRNAs for endogenous normalizationTo identify potential miRNA references for exosomal RNA quantificalion, we analyzed the 173 most abundant known miRNAs from the 192 exosomal RNA sequencing libraries w讓h CV 5%. Based on NormFinder analysis, miR-30e-5p, miR-221-3p, miR-30a-5p, miR-99a-5p, and miR-18lb-5p ranked as the top five most stable miRNAs in ungrouped samples (Fig. 2： Supplementary Table 4). Under Best- Keeper, most miRNAs identif