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miR-539 inhibits prostate cancer progression by directly targeting SPAG5 Zhang, Hongtuan; Li, Shadan; Yang, Xiong; Qiao, Baomin; Zhang, Zhihong; Xu, Yong Apr 1, 2016

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RESEARCH Open AccessmiR-539 inhibits prostate cancerprogression by directly targeting SPAG5Hongtuan Zhang1,2, Shadan Li2,3, Xiong Yang1, Baomin Qiao1, Zhihong Zhang1 and Yong Xu1*AbstractBackground: We conducted multiple microarray datasets analyses from clinical and xenograft tumor tissues tosearch for disease progression-driving oncogenes in prostate cancer (PCa). Sperm-associated antigen 5 (SPAG5)attracted our attention. SPAG5 was recently identified as an oncogene participating in lung cancer and cervicalcancer progression. However, the roles of SPAG5 in PCa progression remain unknown.Methods: SPAG5 expression level in clinical primary PCa, metastatic PCa, castration resistant PCa, neuroendocrinePCa, and normal prostate tissues was investigated. We established multiple in vivo xenografts models using patient-derived tissues and investigated SPAG5 expression trend in these models. We also investigated the functions ofSPAG5 in vivo and in vitro studies. Luciferase reporter assays were performed to investigate potential miRNAs thatcan regulate SPAG5.Results: We identified that SPAG5 expression was gradually increased in PCa progression and its level was significantlyassociated with lymph node metastasis, clinical stage, Gleason score, and biochemical recurrence. Our results indicatedthat SPAG5 knockdown can drastically inhibit PCa cell proliferation, migration, and invasion in vitro and supress tumorgrowth and metastasis in vivo. We identified that miR-539 can directly target SPAG5. Ectopic overexpression of miR-539can drastically inhibit SPAG5 expression and the restoration of SPAG5 expression can reverse the inhibitory effects ofmiR-539 on PCa cell proliferation and metastasis.Conclusion: Our results collectively showed a progression-driving role of SPAG5 in PCa which can be regulated bymiR-539, suggesting that miR-539/SPAG5 can serve as a potential therapeutic target for PCa.Keywords: miRNA, Progression, miR-539, Prostate cancer, SPAG5BackgroundProstate cancer (PCa) is one of the most common andaggressive human malignancies with poor prognosisworldwide [1]. Although considerable advances in ther-apy and diagnosis of PCa have been made, many PCapatients will progress to castration-resistant PCa (CRPC)that is metastatic and shows resistance to hormonaltherapy [2]. Neuroendocrine PCa (NEPC) is anotherlethal form of PCa with most patients dying within 1 yearof diagnosis despite very aggressive chemotherapeuticregimens [3]. Increasing evidence indicates that prostaticadenocarcinoma can undergo a NE transdifferentiationfollowing long-term androgen deprivation therapy, andeventually progress to NEPC [3, 4]. However, NEPCstudies have been hampered by a lack of clinically rele-vant in vivo models of the disease. Recently, our lab suc-cessfully has generated a patient-derived xenograftmodel of complete neuroendocrine transdifferentiation(LTL331R) of a prostate adenocarcinoma (LTL331) atthe Living Tumor Laboratory [5]. Therefore, NEPCxenograft model provides a valuable method for investi-gating the molecular mechanisms of NEPC progression.So discovery of new optimize therapeutic targets formore effective treatment of metastatic PCa, CRPC, andNEPC is urgently needed for improved disease manage-ment and patient survival [6].It is well known that sperm-associated antigen 5(SPAG5) can bind to microtubules as a regulator of thetiming of spindle organization and separation of sisterchromatids [7]. Previous studies indicated that SPAG5* Correspondence: xymnwk@163.com1Department of Urology, National Key Specialty of Urology, Second Hospitalof Tianjin Medical University, Tianjin Key Institute of Urology, Tianjin MedicalUniversity, Tianjin, ChinaFull list of author information is available at the end of the article© 2016 Zhang et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Zhang et al. Journal of Experimental & Clinical Cancer Research  (2016) 35:60 DOI 10.1186/s13046-016-0337-8was an important variable in mitosis and cell cyclecheckpoint regulation [8]. A previous study indicatedthat SPAG5 may act as a promoter in tumorigenesis andprogression [9]. Previous studies showed that SPAG5overexpression can predict poor prognosis in lung can-cer and cervical cancer [9–11] and alter sensitivity totaxol treatment via the mTOR signaling pathway in cer-vical cancer [11]. These results indicated that SPAG5may act as an important oncogene that is involved in theformation and progression of malignancies and mightinfluence the biological behaviors of malignancies. How-ever, the molecular mechanism and role of SPAG5 inPCa development and progression have not yet beeninvestigated.miRNAs are small noncoding RNAs of ~22 nt thatcan regulate gene expression by inhibiting protein trans-lation and promoting the degradation of the targetmRNAs by binding to the 3’ untranslated regions (UTR)of specific mRNAs. An expanding body of evidence hasshowed that the dysregulation of miRNAs is linked tothe development of various types of cancers [12–16]. In-creasing evidence indicates a critical role for miRNAs incancer initiation, promotion, and progression [17–19].In view of the extensive functions of miRNAs andSPAG5, there is an urgent need to identify the molecularmechanism and role of SPAG5 in PCa progression andto investigate whether or not SPAG5 can be targeted bya specific miRNA in PCa.In the current study, we identified that SPAG5 expres-sion level is drastically increased in primary PCa relativeto normal samples, metastatic PCa samples relative to pri-mary PCa, CRPC relative to hormone naïve PCa, andNEPC relative to prostate adenocarcinoma, respectively.We also identified that knockdown of SPAG5 significantlyinhibited the proliferation, migration, and invasion in PCacells. We further confirmed that miR-539 inhibited thePCa growth and metastasis in vivo and also inhibited theproliferation, migration, and invasion in vitro by down-regulating SPAG5 expression. Taken together, ourresearch results position SPAG5 as a progression-drivingoncogene in PCa and a potential therapeutic target inPCa, providing opportunities to explore novel strategiesaimed at reversing PCa progression.MethodsTissue samplesOne hundred eighty PCa and paired adjacent normal tis-sues confirmed by pathologists were collected from thesecond hospital of Tianjin medical university [20, 21].We also collected 20 CRPC tissues. Informed consentwas obtained from each patient, and all of the experi-ments were approved by the ethics committee of theinstitute.PCa cell linesPC-3 (a cell line characteristic of prostatic small cellneuroendocrine carcinoma) and LNCaP cell lines werecultured in RPMI 1640 (Life Technologies, CA) with0.023 IU/ml insulin and 10 % FBS (Invitrogen) in 5 %CO2 cell culture incubator.Plasmid constructionWe amplified a DNA fragment containing the pri-miR-539 gene DNA from genomic human DNA by PCR. Thisfragment was then inserted into pcDNA3 vector. Theprimers for knocking down SPAG5 were synthesized,annealed, and cloned into the pSilencer2.1-neo vector.The SPAG5 cDNA containing the coding sequence wascloned by PCR, and the PCR product was cloned intothe pcDNA3 vector.Colony formation assayWe seed the PCa cells on 35-mm dishes according tothe manufacturers’ instructions. The PCa cells werefixed in methanol, and then stained with crystal accord-ing to the manufacturers’ instructions. Positive colonyformation (>50 cells/colony) would be counted.Migration and invasion assaysThe transwell migration and invasion assays were con-ducted with coated Matrigel (invasion) and uncoated(migration) according to the manufacturers’ instructions.The migrated and invaded cells in the membrane wouldbe fixed, stained, and counted.Luciferase reporter assayThe miR-539-binding site in the SPAG5 3’-UTR region(wild or mutant-type) was cloned downstream of thefirefly luciferase gene in a pGL3-promoter vector. Theluciferase assay was conducted according to the manu-facturers’ instructions.qRT-PCRTotal RNA was extracted using Trizol Reagent accordingto the manufacturers’ instructions. The miR-539 expressionwas measured by TaqMan miRNA assays according to themanufacturers’ instructions, U6 was used for normalization.SPAG5 mRNA expression level was investigated by SYBRgreen qPCR assay and β-actin was used as an endogenouscontrol.Western blotsDetailed procedure for western blot was performed ac-cording to the manufacturers’ instructions. In brief, theinterest protein was resolved by SDS-PAGE and trans-ferred to a PVDF membrane, which was probed withspecific primary antibody against SPAG5 (HPA022479,Sigma). β-actin was used as the endogenous control.Zhang et al. Journal of Experimental & Clinical Cancer Research  (2016) 35:60 Page 2 of 9ImmunohistochemistryThe Immunohistochemistry study was conducted ac-cording to the manufacturers’ instructions using specificprimary antibody against SPAG5 (HPA022479, Sigma).The SPAG5 protein would be classified semiquantita-tively combining the proportion and intensity of posi-tively stained immunoreactive cells [20, 21]. The sum ofthe staining intensity score and the percentage score wasused to define SPAG5 protein level: 0–2, low expressionand 3–4, high expression [20, 21].PCa xenograft researchThe LNCaP and PC-3 cells were stably transfected withpri-miR-539, control, or SPAG5 knockdown vector. TheFig. 1 SPAG5 is gradually increased in normal prostate, primary PCa, metastatic PCa, CRPC, and NEPC. a-e, Scatter plots represent SPAG5 mRNA innormal prostate and primary PCa samples; d-h, and i, Scatter plots represent SPAG5 mRNA in primary PCa and metastatic PCa samples; i, Scatterplots represent SPAG5 mRNA in metastatic PCa and CRPC samples; j. Scatter plots represent SPAG5 mRNA in prostate adernocarcinoma andNEPC samples; k, Scatter plots showed that SPAG5 mRNA was upregulated in three NEPC models (LTL331R, LTL352, and LTL370) relative to theseprostate adenocarcinoma models, metastatic (LTL313H) PCa xenograft line relative to non-metastatic (LTL313B) PCa xenograft line, CRPC xenograftmodel (313BR) compared with LTL313B; l, Scatter plots showed that SPAG5 was downregulated after host castration and significantly upregulatedin fully relapsed NEPC xenograftsZhang et al. Journal of Experimental & Clinical Cancer Research  (2016) 35:60 Page 3 of 9cells were subcutaneously injected into the 6 week-oldnude mice in the flank. Mouse weight and tumor sizewere measured biweekly. The tumor volume was calcu-lated as follows: length × width2 × 1/2. The xenograft tu-mors and cervical lymph nodes samples were collectedand tumor weights were measured 7 weeks after im-plantation. DNA extraction of the cervical lymph nodesand human alu sequence PCR amplification wereperformed to identify the distant metastasis statusaccording to a previous paper [22].Statistical analysisStudent’s t-test was conducted for continuous data.Spearman correlation test was used to investigate the as-sociations between SPAG5 protein level and the clinicaland pathological factors. Survival curves were conductedby the Kaplan-Meier and investigated through log-ranktest. Identified variables were associated with survival bythe Cox proportional hazard regression. A p value lessthan 0.05 was considered significant. Statistical analysiswas performed by SPSS 17.0.ResultsSPAG5 may be a potential progression-driving oncogeneIn order to investigate whether any difference of SPAG5expression exists in primary PCa, metastatic PCa, CRPC,NEPC, and normal prostate tissues, several microarraydatasets were analyzed [23–30]. We identified thatSPAG5 mRNA was significantly overexpressed in pri-mary PCa relative to normal prostate tissues (Fig. 1a-e)and metastatic PCa compared with primary PCa sam-ples, respectively (Fig. 1d-h). The microarray data alsoshowed that SPAG5 mRNA was higher in CRPC samplesthan in hormone naïve PCa samples (Fig. 1i). We alsoinvestigated SPAG5 expression in a clinical cohort thatincluded 30 prostate adenocarcinoma and 7 NEPCpatients. The RNA-seq data of this cohort showed thatSPAG5 was significantly upregulated in NEPC samplescompared with prostate adenocarcinoma samples (Fig. 1j).In order to validate clinical cohort results, we further inves-tigate SPAG5 mRNA expression in a panel of patienttissue-derived PCa xenograft models generated from ourlab. The results indicated that SPAG5 mRNA was upregu-lated in three NEPC models (LTL331R, LTL352, andLTL370) relative to these prostate adenocarcinoma models(Fig. 1k). We also analyzed gene expression profile of pairedmetastatic (LTL313H) and non-metastatic (LTL313B) PCatissue xenograft lines derived from one patient’s primarytumor, LTL313H showed higher expression of SPAG5 rela-tive to LTL313B. Similarly, increased SPAG5 mRNA wasidentified in CRPC xenograft model (313BR) comparedwith LTL313B (Fig. 1k). Our lab also has analysed themicroarray data from LTL331 xenografts before and afterhost castration at various time points. We identified thatSPAG5 was downregulated after host castration and signifi-cantly upregulated in fully relapsed NEPC xenografts(Fig. 1l). Collectively, above data indicated that SPAG5 in-creases gradually during the PCa progression, indicating ithas a very critical role in PCa progression.SPAG5 is significantly associated with PCa progressionand poor prognosisWe also performed microarray analysis to identifywhether SPAG5 was associated with PCa progression[30–33]. We identified that high SPAG5 expression wassignificantly associated with higher PCa grade, andhigher stage (Fig. 2a-e). SPAG5 expression was signifi-cantly overexpressed in dead PCa patients comparedwith living PCa patients at 3 years and 5 years, respect-ively (Fig. 2f, g). We also identified that SPAG5 expres-sion was significantly increased in PCa patients withBCR compared with PCa patients without BCR at 5 yearsand 3 years, respectively (Fig. 2h, i). In order to validatethese results, we further conducted immunohistochemis-try study to investigate SPAG5 protein expression inPCa. SPAG5 protein expression was increased in PCatissues compared with the benign prostate hyperplasia,and the difference was significant. As shown in Fig. 2j-k,the SPAG5 positive staining was localized within thecytoplasm. The analysis indicated weak or no reactivityin less aggressive PCa but strong staining in the aggres-sive PCa and CRPC samples.We investigated the association between SPAG5 pro-tein expression and clinicopathological variables. Asshown in Additional file 1: Table S1, high SPAG5 expres-sion was significantly associated with lymph node metas-tasis, clinical stage, Gleason score, and BCR. We furtheridentified that the high SPAG5 expression patients had ashorter overall or BCR-free survival duration comparedto the patients with low SPAG5 expression. We alsoidentified that SPAG5 was an independent prognosticvariable for BCR-free and overall survival of PCa pa-tients (Additional file 2: Table S2 and Additional file 3:Table S3). Collectively, our data showed high SPAG5protein expression is significantly associated with PCaprogression and poor prognosis.SPAG5 silence inhibits PCa cell colony formation,migration, and invasionTo investigate the functional significance of SPAG5overexpression in PCa, we reduced SPAG5 expression inPCa cells and studied its impacts on cell colony forma-tion, migration, and invasion. We employed stableknockdown strategy targeting SPAG5 in PCa cells. Weidentified significant decrease in colony formation inSPAG5 silence cells relative to control cells (Additionalfile 4: Figure S1A). We also identified that SPAG5silence significantly inhibited PC-3 and LNCaP cellZhang et al. Journal of Experimental & Clinical Cancer Research  (2016) 35:60 Page 4 of 9migration and invasion (Additional file 4: Figure S1B, C).We confirmed that SPAG5 protein was significantly re-duced in SPAG5 silence cells compared with controlcells (Additional file 4: Figure S1D).SPAG5 is a direct target of miR-539 which may be apotential anti-progression miRNAWe employed four miRNA target prediction algorithmsprovided by miRanda, miRWalk, PICTAR5, and Targets-can. After integrating the results, we select miR-539 forfurther research because of its tumor suppressorproperties in osteosarcoma and thyroid cancer [34, 35].However, up to now, its roles in PCa and other cancersare still unclear. We identified that miR-539 is signifi-cantly decreased in both primary PCa compared withnormal prostate tissues and metastatic PCa relative toprimary PCa tissues (Additional file 5: Figure S2) [30].The binding site for miR-539 at 3’-UTR of SPAG5 wasdepicted (Fig. 3a). We found that co-transfection withmiR-539 can inhibit the luciferase activity when theconstruct contained the 3’UTR of SPAG5 (Fig. 3b). Mu-tation of the binding site can reverse the effects. TakenFig. 2 SPAG5 overexpression is associated with PCa progression and poor prognosis. a-d, Box plots showed that SPAG5 was significantly increased inpatients with higher Gleason score relative to patients with lower Gleason score; e, Box plots showed that SPAG5 was significantly increased in patientswith higher grade relative to patients with lower grade; f, g, Box plots showed that SPAG5 mRNA was significantly overexpressed in dead PCa patientscompared with living PCa patients at 3 years and 5 years, respectively; h, i, Box plots showed that SPAG5 mRNA was significantly increased in PCa patientswith BCR compared with PCa patients without BCR at 3 years, and 5 years, respectively. j-l, Immunohistochemical analysis of SPAG5 in, less aggressive PCa(j), aggressive PCa (h), and CRPC (l)Zhang et al. Journal of Experimental & Clinical Cancer Research  (2016) 35:60 Page 5 of 9together, above results indicated that SPAG5 was a dir-ect target of miR-539. We further performed qRT-PCRand western blot to study whether ectopic overexpres-sion of miR-539 can change SPAG5 expression. Weidentified that SPAG5 mRNA expression was signifi-cantly downregulated in miR-539 transfectants as com-pared with control cells (Fig. 3c). We also identified thatSPAG5 mRNA expression was inversely associated withthe miR-539 expression level (Fig. 3d). Overall, these re-sults showed that miR-539 is gradually decreased in PCaprogression and may directly target SPAG5.miR-539 inhibits PCa cell proliferation, invasion, andmigration by directly targeting SPAG5We investigated whether miR-539 can change the effectsof SPAG5 on PCa cell colony formation, invasion, andmigration. We found that SPAG5 overexpression cansignificantly abrogate the inhibitory effects of PCa cellcolony formation induced by miR-539 (Additional file 4:Figure S1A). We also found that SPAG5 overexpressioncan reverse the inhibitory effects of PCa cell migrationand invasion induced by miR-539 (Additional file 4:Figure S1B, C). The efficiency of miR-539 upregulationwas studied by western blots (Additional file 4: FigureS1D). We found that SPAG5 knockdown can result insimilar effects induced by miR-539 upregulation in PCacells (Additional file 4: Figure S1).SPAG5 plays a critical role in PCa growth and metastasisin vivoTo study the role of SPAG5 on PCa growth and metas-tasis in vivo, a murine xenograft model using stableSPAG5 silence PCa cells was employed. SPAG5 silencecan lead to significantly reduced tumor volume andweight relative to control mice (Fig. 4a-d). SPAG5 si-lence tumor tissues showed reduced staining for SPAG5(Fig. 4e-g). SPAG5 silence significantly inhibited PC-3cell’s ability to metastasize relative to control group(Fig. 4h). miR-539 upregulation can result in attenuatedmetastasis relative to the control group, and lead tosimilar results caused by SPAG5 knockdown (Fig. 4). Wedid not find any metastases in control or experimentalLNCaP xenografts. Collectively, above data suggestedthat SPAG5 may play a critical role in PCa growth andmetastasis in vivo.DiscussionIt is well known that surgery and radiation therapy cancure the localized PCa [36, 37]. However, many treatedPCa patients will experience local recurrence or metastasis[2–4]. Metastatic PCa, CRPC, and NEPC are highly resist-ant to conventional therapy and are at present incurable[36, 38, 39]. We know that most NEPC patients resultfrom transdifferentiation of a typical prostate adenocarcin-oma following androgen-deprivation therapy. We haveFig. 3 SPAG5 is a direct target of miR-539 in PCa. a, Computational analysis indicating that miR-539 potentially targeted SPAG5. b, Relative luciferaseactivities were studied in PCa cells. c, Decrease in SPAG5 mRNA expression by miR-539 was investigated using qRT-PCR. d, Decrease in SPAG5 proteinexpression by miR-539 was investigated using western blots. e, SPAG5 mRNA was inversely associated with miR-539 in 180 pairs of PCa tissuesZhang et al. Journal of Experimental & Clinical Cancer Research  (2016) 35:60 Page 6 of 9recently established the first in vivo model of completeneuroendocrine transdifferentiation using patient-derivedxenografts. Development of therapeutic approaches spe-cifically targeting metastatic PCa, CRPC, and NEPC asso-ciated genes may lead to improved PCa management.Previous study reported overexpression of SPAG5 in sev-eral types of cancers [9–11]. However, there has been noreport about SPAG5 functional analyses in PCa. In currentstudy, we try our best to investigate SPAG5 expressionpattern and its association with PCa carcinogenesis,progression, and prognosis. Our data strongly indicatesthat SPAG5 functions as a progression-driving oncogenein PCa.We first found a significant upregulation of SPAG5expression in primary PCa relative to normal prostatetissues, metastatic PCa relative to primary PCa, CRPCrelative to hormone naïve PCa, and NEPC relative toprostate adenocarcinoma through microarray analysis,Fig. 4 SPAG5 plays a critical role in PCa growth and metastasis in vivo. a, b, SPAG5 knockdown in PCa cells significantly inhibits tumor growth ina mouse xenograft model; c, d, Tumor weights of corresponding mouse xenograft models; e-g, SPAG5 expression analysis was conducted at proteinlevel by immunohistochemical analysis and western blot. h, SPAG5 played an important role in PCa metastasis in vivo. Ectopic expression of miR-539can mimic the effects induced by SPAG5 knockdown. All * indicated P value less than 0.05Zhang et al. Journal of Experimental & Clinical Cancer Research  (2016) 35:60 Page 7 of 9indicating SPAG5 may has a critical role in PCa progres-sion. In order to find the potential critical role of SPAG5in PCa, we further studied the association of SPAG5protein staining with clinicopathological variables inPCa. Results showed that SPAG5 positive staining wassignificantly associated with PCa progression. Resultsalso suggested that positive staining of SPAG5 was inde-pendently associated with unfavorable outcome of PCapatients. The prognostic value of SPAG5 was statisticallysignificant in multivariate analysis adjusted for signifi-cant variables from univariate analysis, which suggestedSPAG5 expression may be a good molecular marker topredict PCa prognosis. This is the first direct evidence ofthe association between SPAG5 and clinicopathologicalvariables of PCa and the prognostic role of SPAG5 inPCa.In support of our observations in clinical research, weidentified important functional roles of SPAG5 in PCacells. Besides the reduction of PCa cell colony formation,we also found impaired migration and invasion abilitiesof PC-3 and LNCaP cells upon inducible shRNA-mediated SPAG5 silence, indicating that the SPAG5functioned as a progression associated gene in PCa. Wefurther confirmed that SPAG5 knockdown can signifi-cantly inhibit tumor growth and metastasis in vivo. In-creasing evidence showed miRNAs have critical roles inseveral biological processes, including differentiation,progression, angiogenesis, proliferation, migration, andinvasion. We employed 4 public miRNA target predic-tion algorithms to identify potential miRNAs which canregulate SPAG5. Among of these potential miRNAs,miR-539 attracted our attention due to its tumor sup-pressor role in osteosarcoma and thyroid cancer [34],and its unclear roles in PCa and other cancers. The datafrom the Taylor et al. study confirmed that miR-539 ex-pression is low in the more advanced primary PCa, withthe lowest expression in metastatic tissues. We furtheridentified that miR-539 level was inversely associatedwith SPAG5 mRNA level. Our results further confirmedthat the miR-539 can directly target SPAG5. As expect-edly, ectopic expression of miR-539 can result in similarresults caused by SPAG5 silence in PCa cells. Further-more, ectopic overexpression of SPAG5 can significantlyreverse the inhibitory effects of miR-539. Taken together,above results indicated that miR-539 can supress PCacell colony formation, migration, and invasion by dir-ectly targeting SPAG5.We further investigated the role of SPAG5 on PCagrowth and metastasis in vivo using a murine xenograftmodel. We found that SPAG5 knockdown can contrib-ute to decreased xenograft tumor volume and weightrelative to control group. We also found that SPAG5knockdown can inhibit PC-3 cell’s ability to metastasizerelative to control group. As expected, miR-539overexpression can lead to attenuated metastasis relativeto the control group, and lead to similar results inducedby SPAG5 knockdown. Taken together, above results in-dicated that SPAG5 may play a critical role in PCagrowth and metastasis in vivo.ConclusionsTo our best knowledge, this is the first study to report thepotential progression-driving function of SPAG5 in PCa.Our study also provides first experimental evidence thatmiR-539 can inhibit PCa cell growth and metastasis inPCa by directly repressing SPAG5. The newly identifiedmiR-539/SPAG5 signal pathway may provide new insightsinto the progression of PCa and a potential therapeutictarget for PCa treatment. Overall, above results indicatedthat SPAG5 may play a progression-driving role in PCaand could serve as a viable therapeutic target.Additional filesAdditional file 1: Table S1. Clinicopathologic factors and SPAG5 proteinexpression in 180 PCa patients. (DOC 68 kb)Additional file 2: Table S2. Prognostic value of SPAG5 protein expressionfor the biochemical recurrence free survival in univariate and multivariateanalyses by Cox regression. (DOC 34 kb)Additional file 3: Table S3. Prognostic value of SPAG5 protein expressionfor the overall survival in univariate and multivariate analyses by Coxregression. (DOC 34 kb)Additional file 4: Figure S1. miR-539 inhibits PCa cell proliferation,migration and invasion by targeting SPAG5 in vitro. A, Ectopic expressionof miR-539 can mimic the suppression of colony formation induced bySPAG5 knockdown in PC-3 and LNCaP cells; B, Ectopic expression of miR-539 can mimic the suppression of migration activity induced by SPAG5knockdown in PC-3 and LNCaP cells; C, Ectopic expression of miR-539can mimic the suppression of invasion activity induced by SPAG5 knockdownin PC-3 and LNCaP cells; D, The efficiency of SPAG5 knockdown and ectopicexpression of miR-539 was confirmed at protein level by western blot.(JPG 145 kb)Additional file 5: Figure S2. miR-539 is downexpressed in primary PCaand metastatic PCa. miR-539 level was gradually decreased in normalprostate, primary PCa, and metastatic PCa samples. (JPG 173 kb)AbbreviationsBCR: biochemical recurrence; CI: confidence interval; CRPC: castration-resistantprostate cancer; NEPC: neuroendocrine prostate cancer; PCa: prostate cancer;PSA: prostate-specific antigen; SPAG5: sperm-associated antigen 5.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsZH, LS, QB and XY conceived and designed the project, performed theexperiments and wrote the manuscript. ZH, and XY contributed to thewriting and to the critical reading of the paper. ZH, and QB performedpatient collection and clinical data interpretation. ZH, and YX participatedperformed the statistical analysis. All authors read and approved the finalmanuscript. All authors read and gave their approval for the final version ofthe manuscriptAcknowledgementsThis study was supported by the National Natural Science Foundation ofChina (NO: 81502220), and Science Foundation of Tianjin medical university(NO: 2009GSI18).Zhang et al. 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