BZW1 Promotes Cell Proliferation in Prostate Cancer by Regulating TGF-β1/Smad Pathway

BZW1 Promotes Cell Proliferation in Prostate Cancer by Regulating TGF-β1/Smad Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article BZW1 Promotes Cell Proliferation in Prostate Cancer by Regulating TGF-β1/Smad Pathway Teng-Cheng Li, Zhuo-Lun Sun, Chu-Tian Xiao, Jie-Ying Wu, Ke Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-45376/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Recently, basic leucine zipper and the W2 domain-containing protein 1 (BZW1) is reported to be implicated in tumor progression. However, the role of BZW1 in prostate cancer remains unknown. This study is aimed to investigate the expression of BZW1 and its influence on cell proliferation in prostate cancer. Methods The expression levels of BZW1 were measured in 136 cases of prostate cancer and matched adjacent non-cancerous prostate tissues by quantificational real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). Then, the effect of BZW1 on cell proliferation was further explored. Results QRT-PCR analysis shown that the mRNA levels of BZW1 in prostate cancer were significantly greater compared with those in matched adjacent non-cancerous prostate tissues ( P <0.001). IHC results shown the high-expression rate of BZW1 in prostate cancer and matched adjacent non-cancerous prostate tissues were 68.4% and 32.4%, and the difference was statistically significant ( P <0.001). BZW1 high-expression significantly correlated with T stage, lymph node metastasis, prostate specific antigen (PSA) and Gleason score ( P <0.05). Patients with BZW1 high-expression presented unfavorable prognosis compared with those with BZW1 low-expression ( P =0.002). In addition, CCK-8 and Colony formation assays revealed that BZW1 over-expression significantly promoted cell proliferation in vitro . Tumor xenograft shown BZW1 knockdown significantly inhibited tumor growth in vivo. Moreover, BZW1 overexpression activated the TGF-β1/Smad1/Smad3 pathway. Conclusion BZW1 over-expression predicts poorer prognosis and promotes cell proliferation in prostate cancer by regulating TGF-β1/Smad pathway. Cancer Biology Oncology BZW1 prognosis Prostate cancer survival biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Prostate cancer (PCa) is one of the most prevalent urological cancers in men worldwide [1–3]. The incidence of PCa is increased worldwide in recent years [4–5]. In the USA and Europe, PCa accounts for the second most leading cause of cancer-related mortality [6–7]. Although the testing of PSA was generally used in the early diagnosis, the incidence and mortality rate of PCa are still increasing [8–10]. Therefore, identifying new potential markers of prostate cancer is crucial for the early diagnosis and clinical therapy. BZW1 is a member of the bZIP superfamily of transcription factors, which contains C-terminal W2-type HEAT domains [11–12]. BZW1 plays a key role in the cell cycle by regulating histone H4 gene transcription [13]. Moreover, BZW1 promotes cell proliferation in salivary mucoepidermoid carcinoma and lung adenocarcinoma [14–16]. BZW1 overexpression is an independent prognostic marker in lung adenocarcinoma [16]. However, the biological function of BZW1 in prostate cancer remains unknown. Here, the expression of BZW1 and its clinical significance in prostate cancer were investigated. Moreover, the effect of BZW1 expression on cell proliferation and its potential mechanism were explored. Methods Patients and samples A total of 136 prostate cancer and matched adjacent non-cancerous prostate tissues were enrolled in this study. All patients received surgery resection during 2010 to 2018, while no patient treated with chemotherapy or radiotherapy before surgery. Clinicalpathological characters were obtained from hospitalized records. The follow-up time was ranged from 8 to 64 months. This study was supported by the ethics committee of Sun Yat-sen University (No. Z81771573) and performed in accordance with the Declaration of Helsinki. The informed consent was signed by each patient. Cell Culture And Transfection Human prostate cancer cell lines (PC3, Du145 and LnCap) were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were cultured with RPMI-1640 (Gibco) with 10% heat-inactivated fetal bovine serum (FBS, Hyclone) at 37 °C in a humidified incubator with 5% CO 2 . Cell transfection was performed by Lipofectamine™ 2000 (Invitrogen, Thermo Fisher Scientific, Inc, USA) according to the manufacturer's instructions. Then, cells were cultured with 10% heat-inactivated fetal bovine serum (FBS, Hyclone) supplemented with puromycin (0.5 µg/mL). BZW1 down-regulation was performed by transferring shRNA lentivirus vectors (Genepharma, Suzhou, China) and BZW1 over-expression was performed by transferring recombinant lentivirus vectors (Genepharma, Suzhou, China). Non-target shRNA (Genepharma, Suzhou, China) lentivirus vectors were used as the negative control (NC). QRT-PCR Total RNA was extracted from fresh tissues and cells by using TRIzol Reagent (Life Technologies). According to the manufacture’s protocol, the RNA was reversed into cDNA using a PrimeScript RT kit (Takara, Dalian, China). Real-time quantitative PCR was performed by SYBR1 Premix Ex Taq (DDR041A, TaKaRa, Dalian, China). Primers for BZW1 were 5′-ACTGGTGTTCTTCTGGCTAA-3′ (Forward) and 5′-GTGCTCATTACACTTGACCA-3′ (Reverse). The primer sequence for GAPDH were 5′-CTGAACGGGAAGCTCACTGG-3′ (Forward) and 5′-TGAGGTCCACCACCCTGTTG-3′ (Reverse). GAPDH was considered as the internal control and each test was repeated three times. The relative expression level of target genes was calculated using the 2 −ΔΔCt method. Western Blotting Proteins were extracted by radioimmunoprecipitation assay (RIPA) Lysis buffer (Abcam, Cambridge, MA, USA) and quantified by bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). 40 µg of protein per sample was separated onto 10% SDS-PAGE gels and transferred onto polyvinyl difluoride (PVDF) filter membrane (Bio-Rad, Hercules, CA, USA). Then, PVDF membranes were blocked with 5% non-fat dried milk for 2 h, and incubated with anti-BZW1 (1: 1000, ABcam Corp, USA), anti-Smad1 (1: 1000, ABcam Corp, USA), anti-p-Smad1 (1: 1000, ABcam Corp, USA), anti-Smad3 (1: 1000, ABcam Corp, USA), anti-p-Smad3 (1: 1000, ABcam Corp, USA), anti-TGF-β1 (1: 2000, ABcam Corp, USA) and anti-GAPDH (1: 2000, ABcam Corp, USA), respectively at 4 ℃ overnight. Finally, membranes were incubated with secondary antibodies (horseradish peroxidase-conjugated anti-rabbit). Signals were developed using an enhanced chemiluminescence reaction kit (Applygen Technologies, Beijing, China). Immunohistochemistry (ihc) Tissue sections (Two-micrometre-thick) were deparaffinized with xylene and alcohol. Antigen retrieval was completed by sodium citrate buffer (pH 6.0). Then, sections were incubated with rabbit polyclonal BZW1 antibody (Abcam Corp, USA) (diluted 1: 100) overnight at 4 °C. Sections were washed with Phosphate-Buffered Saline (PBS) and incubated with biotin-labeled secondary antibodies for 30 minutes at room temperature. Signals were detected by diaminobenzidine tetrahydrochloride (DAB). PBS substituted with primary antibody was regarded as negative controls. The positive controls were performed by lung adenocarcinoma with positive expression of BZW1 [15]. The scores were calculated by the number of positive cells and staining intensity. Percentage of cells was recorded as 1 ( 51%). Staining intensity was recorded as blank (0), weak (1, +), moderate (2, ++) and strong (3, +++). Total score of each section was ranged from 0 to 9. Based on the semiquantitative analysis, BZW1 expression was scored as low-expression (< 4) and high-expression (≥ 4) [15]. Cell counting kit-8 (CCK-8) analysis CCK-8 assay was used to evaluate the cell proliferation. Cells were transferred and seeded in 96-well plates (1.0 × 10 3 cell/well) for 5 days. Then, cells were cultured with 10 µL of CCK-8 (Dojindo, Kumamoto, Japan) for 3 h, and the optical density of each well was examined by a microplate reader (BioTek, Winooski, VT, USA) at 450 nm. Each test was repeated in triplicate at the same condition. Colony formation analysis Tansfected cells (1.0 × 10 3 cell/well) were seeded into 6-well plates. After 2 weeks, cells were obtained and fixed with 100% methanol. Then, cells were incubated with 0.1% crystal violet and stained with hemocytometer for counting. Each experiment was repeated in triplicate at the same condition. Tumor Xenograft In Vivo Balb/c nude mice (4 weeks old) were purchased from Animal Center and kept on specific pathogen-free conditions in accordance with institutional guidelines. To evaluate the effect of BZW1 on tumor growth, 6 × 10 6 prostate cancer cells with shRNA lentivirus vectors were subcutaneously injected into the flank regions of legs (5 mice per group). Negative controls (NC) were treated with prostate cancer cells with non-target shRNA lentivirus vectors. After 2 weeks, tumor size and tumor weight were calculated. Statistical analysis All data was characterized as mean ± standard deviation, and analyzed by SPSS software (version 19.0; SPSS, Chicago, IL, USA). The mRNA expression levels of BZW1 between prostate cancer and matched adjacent non-cancerous tissues were analyzed by paired t -test. The correlation between BZW1 expression and clinicalpathological characters was evaluated by Chi-square test (χ 2 ). Survival analysis was performed by using the Kaplan-Meier method with log-rank test. Hazard Ratio (HR) was evaluated using the Cox’s proportional hazards model. P < 0.05 was considered as statistical significance. Results BZW1 is highly expressed in prostate cancer and correlated with clinicalpathological characters To evaluate the expression of BZW1, the mRNA levels of BZW1 were detected in 136 cases of prostate cancer and matched adjacent non-cancerous prostate tissues by qRT-PCR. Results shown the mRNA levels of BZW1 was significantly greater in prostate cancer compared with those in matched adjacent non-cancerous prostate tissues (Fig. 1 A, P < 0.001). Then, the protein expression levels of BZW1 was detected by IHC. As shown in Fig. 1 B-E, positive staining of BZW1 was mainly located in the cell cytoplasm as brown and yellow. Semiquantitative analysis shown that the high-expression rate of BZW1 in prostate cancer and non-cancerous prostate tissues were 68.4% and 32.4%, and the difference was statistically significant (Table 1 , P < 0.001). Table 1 BZW1 expression was detected in PCa and non-cancerous prostate tissues by IHC Types N BZW1 P value low-expression high-expression PCa tissues 136 43 (31.6) 93 (68.4) < 0.001 Non-cancerous prostate tissues 136 92 (67.6) 44 (32.4) To investigate the clinical significance of BZW1 in prostate cancer, the correlation between BZW1 expression and clinicalpathological characters was investigated. Results shown BZW1 high-expression was significantly correlated with T stage, lymph node metastasis, PSA and Gleason score ( P 0.05, Table 2 ). Furthermore, Kaplan-Meier analysis revealed that patients with BZW1 high-expression presented unfavorable prognosis than those with BZW1 low-expression (Fig. 2 , P = 0.002). T stage, lymph node metastasis, PSA and Gleason score rather than age and smoke history were significantly correlated with overall survival time ( P < 0.05, Table 3 ). Moreover, multivariate Cox regression analysis shown that BZW1 expression, T stage, lymph node metastasis, PSA and Gleason score were independent prognostic factors for overall survival in prostate cancer (Table 4 , P 60 76 19 57 Smoke history Negative 79 29 40 0.058 Positive 57 14 43 T stage T1-T2 65 34 31 < 0.001 T3 71 9 62 Lymph node metastasis Negative 70 33 37 < 0.001 Positive 66 10 56 PSA ( ng/ml) < 10 34 18 16 0.003 ≥ 10 102 25 77 Gleason Score < 8 73 34 39 < 0.001 ≥ 8 63 9 54 Table 3 Survival analysis was performed by Kaplan-Meier method Variables N Mean survival time (Month, 95% CI) P value BZW1 expression Low-expression 43 56 (51–61) 0.002 High-expression 93 44 (40–48) Age (years) ≤ 60 60 48 (43–53) 0.969 > 60 76 47 (42–52) Smoke history Negative 79 47 (42–52) 0.439 Positive 57 49 (44–54) T stage T1-T2 65 55 (51–59) < 0.001 T3 71 41 (37–45) Lymph node metastasis Negative 70 54 (50–58) < 0.001 Positive 66 41 (37–45) PSA (ng/ml) < 10 34 55 (49–61) 0.024 ≥ 10 102 45 (41–49) Gleason Score < 8 73 55 (51–59) < 0.001 ≥ 8 63 38 (34–42) Table 4 Prognostic factors were analyzed by Cox’s proportional hazards model Variables Hazard rate 95% CI P value BZW1 (High-expression VS Low-expression) 3.394 1.658–6.947 0.001 Age (≤ 60 years VS > 60 years) 0.686 0.385–1.223 0.202 Smoke history (Positive VS Negative) 0.582 0.327–1.039 0.067 T stage (T3 VS T1-T2) 2.878 1.614–5.132 < 0.001 PSA (≥ 10 ng/ml VS < 10 ng/ml ) 2.096 1.056–4.163 0.034 Lymph node metastasis (Positive VS Negative) 2.636 1.516–4.584 0.001 Gleason Score (≥ 8 VS < 8) 3.235 1.857–5.635 < 0.001 BZW1 promotes cell proliferation in prostate cancer by regulating TGF-β1/Smad pathway To explore the biological functions of BZW1 in prostate cancer, the influence of BZW1 expression on cell proliferation was investigated. As shown in Fig. 3 A, BZW1 was highly expressed in PC3 cells, but was relatively lower in DU145 and LnCap cells. Thus, BZW1 expression was knocked down in PC3 cells and over-expressed in LnCap cells. QRT-PCR and Western blot analysis shown BZW1 down-regulation and up-regulation were successfully performed (Fig. 3 B and 3 C). Then, cell proliferation was evaluated by CCK-8 and Colony formation assays in vitro . CCK-8 assay showed that BZW1 knockdown significantly inhibited the proliferation in PC3 cells (Fig. 3 D), while BZW1 over-expression significantly promoted the proliferation in LnCap cells. The colony number in cells with BZW1 down-regulation was significantly decreased, while was significantly increased in cells with BZW1 over-expression (Fig. 4 A). To further investigate the effect of BZW1 expression on cell proliferation, xenografts in vivo was performed. Results showed that the size and weight of tumor with BZW1 knockdown were significantly decreased compared with those in NC groups (Fig. 4 B and 4 C). To explore the potential mechanism of BZW1, TGF-β1/Smad pathway was investigated. As shown in Fig. 4 D, the expression of TGF-β1 was significantly decreased with the knockdown of BZW1, while was significantly up-regulated by BZW1 over-expression. The protein expression levels of Smad1 and Smad3 were unchanged regardless of whether the BZW1 expression interference, while BZW1 down-regulation resulted in the decrease of p-Smad1 and p-Smad3. Discussion BZW1, as one of transcription factors, encode a N-terminal bZIP domain and a C-terminal nucleotide binding domain [11–12]. The proteins and transcription factors of bZIP domain play crucial roles in cancer development [17–18]. Recently, BZW1 over-expression was observed in mucoepidermoid carcinoma [14] and lung adenocarcinoma [16]. However, the role of BZW1 in prostate cancer remains unknown. Here, the expression and clinical significance of BZW1 was investigated in prostate cancer. Results shown that the mRNA and protein levels of BZW1 in prostate cancer were significantly higher compared with those in non-cancerous tissues. These data indicated that BZW1 overexpression was associated with the formation of prostate cancer, which was consistent with the observations in mucoepidermoid carcinoma [14] and lung adenocarcinoma [16]. BZW1 over-expression might be helpful for the diagnosis of prostate cancer. In addition, BZW1 over-expression significantly correlated with T stage, lymph node metastasis, PSA and Gleason score, suggesting that BZW1 over-expression was connected with the progression of PCa. Moreover, survival analysis shown that BZW1 as well as T stage, lymph node metastasis, PSA and Gleason score were associated with patients’ survival, which could be as independent prognostic factors in PCa. Moreover, Chiou et al [16] reported BZW1 over-expression was an independent poor prognosis marker in lung adenocarcinoma. Subsequently, the influence of BZW1 expression on cell proliferation and its potential mechanism were explored. Results shown BZW1 down-regulation significantly suppressed cell proliferation, while BZW1 over-expression significantly promoted cell proliferation in vitro . Xenografts assays further validated that BZW1 knockdown significantly inhibited the proliferation of prostate cancer in vivo . To explore the potential mechanism of BZW1, TGF-β1/Smad pathway was investigated. It is well known that TGF-β/Smad pathway is involved in cell proliferation [19–21]. Results shown that BZW1 over-expression activated the TGF-β1/Smad pathway. Thus, these data indicated that BZW1 over-expression promoted cell proliferation, which was connected with the TGF-β1/Smad pathway. Conclusion These data indicate that BZW1 is highly expressed in prostate cancer and correlates with tumor progression and prognosis. Moreover, BZW1 overexpression promotes cell proliferation by regulating TGF-β1/Smad pathway. Declarations Not applicable. Declarations Authors’ contributions KL conceived and designed the study. TCL and ZLS performed the analysis. JYW and CTX coordinated the statistical analyses of results. KL and TCL wrote the manuscript. All authors read and approved the manuscript. All authors consent for publication. Funding This study is supported by the Pearl River S&T Nova Program of Guangzhou (No. 201710010039), Basic Service Charge Young Teachers Cultivation Project of Sun Yat-sen University (17ykpy48) and National Natural Science Foundation (No. 81771573). The design of the study, collection of samples, analysis of results, interpretation of data, and writing of the manuscript was done by the authors (for details on authors’ contribution see below). Availability of data and materials The datasets used and analyzed during this study are available from the corresponding author on reasonable request. Ethics approval and consent to participate This study was reviewed and approved by the internal review boards (IRB) of the involved institutions: Sun Yat-sen University. (No. Z81771573). All eligible subjects signed an informed consent to participate in this study. Consent for publication Not applicable. Competing interests All author declare they have no financial disclosures and conflicts of interest. References 1. Li Z, Wei D, Zhu C, Zhang Q. Effect of a patient education and rehabilitation program on anxiety, depression and quality of life in muscle invasive Prostate cancer patients treated with adjuvant chemotherapy. Medicine (Baltimore). 2019, 98(44): e17437. 2. Zhao F, Yu X, Xu M, Ye S, Zang S, Zhong W, Ren G, Chen X, Yan S. 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Bi JG, Zheng JF, Li Q, Bao SY, Yu XF, Xu P, Liao CX. MicroRNA-181a-5p suppresses cell proliferation by targeting Egr1 and inhibiting Egr1/TGF-β/Smad pathway in hepatocellular carcinoma. Int J Biochem Cell Biol. 2019, 106: 107–116. 21. Romero D, Kawano Y, Bengoa N, Walker MM, Maltry N, Niehrs C, Waxman J, Kypta R. Downregulation of Dickkopf-3 disrupts prostate acinar morphogenesis through TGF-β/Smad signalling. J Cell Sci. 2013, 126(Pt 8): 1858–1867. Supplementary Files supplementarymaterial.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-45376","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":1235681,"identity":"83ba15fb-9e3e-41a3-b847-eee7ad059a78","order_by":0,"name":"Teng-Cheng Li","email":"","orcid":"","institution":"sun yat-sen university","correspondingAuthor":false,"prefix":"","firstName":"Teng-Cheng","middleName":"","lastName":"Li","suffix":""},{"id":1235682,"identity":"44e096ea-e2ef-4e95-9555-21003aac9e7d","order_by":1,"name":"Zhuo-Lun Sun","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Zhuo-Lun","middleName":"","lastName":"Sun","suffix":""},{"id":1235683,"identity":"5f373754-49a6-40ce-bc38-918428f82044","order_by":2,"name":"Chu-Tian Xiao","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Chu-Tian","middleName":"","lastName":"Xiao","suffix":""},{"id":1235684,"identity":"0a256914-3d52-496d-b652-738dd9b0fac0","order_by":3,"name":"Jie-Ying Wu","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Jie-Ying","middleName":"","lastName":"Wu","suffix":""},{"id":1235685,"identity":"e2be104a-9d5c-44cc-b54b-1273d81aba11","order_by":4,"name":"Ke Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIie3QsQqDQAyA4UjguqS6nuDQR0gRnIQ+SBdd3Lo7OAgFO3bt4xwEbhJ8BX0DH6BDXTuUpluH++b8JHcAQfCHYkTnVu4oQZRZlRg09fJofZbeTMO6BCjPacSSJzpYXbKDwu4HQ7kQMHTlWXEYNDYdMipk72bwzaVXJN4ety2FxBVHvWiSaLD1gJRfia0yQWQ3IjHqExMtfevJyvbJleYtSTKt8uTulNxF5rUrvyfvqt/GgyAIgk9eHAM1pik/ARwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4918-6523","institution":"Sun Yat-Sen University","correspondingAuthor":true,"prefix":"","firstName":"Ke","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2020-07-18 11:08:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-45376/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-45376/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":1836907,"identity":"45d92440-badd-41da-a118-e78d89cfac3f","added_by":"auto","created_at":"2020-08-07 14:49:32","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":190659,"visible":true,"origin":"","legend":"The expression levels of BZW1 were detected in prostate cancer. A: qRT-PCR revealed that the mRNA levels of BZW1 were higher in PCa compared with those in NC. (NC: non-cancerous prostate tissues). B: BZW1 low-expression in non-cancerous prostate tissues by IHC (Score=0). C: BZW1 low-expression in PCa by IHC (Score=2). D: BZW1 high-expression in PCa by IHC (Score=4). E: BZW1 high-expression in PCa by IHC (Score=6). ","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-45376/v1/Figure1.jpg"},{"id":1836908,"identity":"1e265c5c-3fb9-4f04-b8e4-480bd8d0465d","added_by":"auto","created_at":"2020-08-07 14:49:32","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":72396,"visible":true,"origin":"","legend":"Kaplan-Meier survival analysis shown patients with BZW1 over-expression had unfavorable survival.","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-45376/v1/Figure2.jpg"},{"id":1836909,"identity":"b3c84251-dfde-410c-afc3-d145b4a00fc9","added_by":"auto","created_at":"2020-08-07 14:49:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":160691,"visible":true,"origin":"","legend":"BZW1 overexpression promoted cell proliferation in vitro. A: The protein expression of BZW1 was detected in prostate cancer cell lines by Western blot. B: qRT-PCR analysis revealed that BZW1 down-regulation and up-regulation were successfully performed. C: Western blot analysis revealed that BZW1 down-regulation and up-regulation were successfully performed D: CCK-8 assay shown BZW1 down-regulation significantly inhibited cell proliferation, while BZW1 overexpression significantly promoted cell proliferation. (Full-length blots/gels are presented in Supplementary Figure)","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-45376/v1/Figure3.jpg"},{"id":1836910,"identity":"72c25236-0e42-4729-aa78-db9a9d84a864","added_by":"auto","created_at":"2020-08-07 14:49:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":165632,"visible":true,"origin":"","legend":"BZW1 overexpression promoted cell proliferation in vivo. A: BZW1 overexpression promoted colony formation. B: Xenografts assay indicated tumor size was decreased by down-regulating the expression of BZW1. C：Xenografts assay indicated tumor weight was significantly reduced by down-regulating the expression of BZW1. D: BZW1 over-expression activated the TGF-β/Smad pathway. (Full-length blots/gels are presented in Supplementary Figure)","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-45376/v1/Figure4.jpg"},{"id":13569588,"identity":"78c621c7-e9aa-4694-be52-cbe99ac421f4","added_by":"auto","created_at":"2021-09-17 03:40:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":778495,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-45376/v1/b0720d42-56c8-4ece-a342-32e25de812b2.pdf"},{"id":1836912,"identity":"62cb62c5-b715-4c56-80d4-6a9548555706","added_by":"auto","created_at":"2020-08-07 14:49:33","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":145146,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterial.doc","url":"https://assets-eu.researchsquare.com/files/rs-45376/v1/supplementarymaterial.doc"}],"financialInterests":"","formattedTitle":"\u003cp\u003eBZW1 Promotes Cell Proliferation in Prostate Cancer by Regulating TGF-β1/Smad Pathway\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eProstate cancer (PCa) is one of the most prevalent urological cancers in men worldwide [1\u0026ndash;3]. The incidence of PCa is increased worldwide in recent years [4\u0026ndash;5]. In the USA and Europe, PCa accounts for the second most leading cause of cancer-related mortality [6\u0026ndash;7]. Although the testing of PSA was generally used in the early diagnosis, the incidence and mortality rate of PCa are still increasing [8\u0026ndash;10]. Therefore, identifying new potential markers of prostate cancer is crucial for the early diagnosis and clinical therapy.\u003c/p\u003e \u003cp\u003eBZW1 is a member of the bZIP superfamily of transcription factors, which contains C-terminal W2-type HEAT domains [11\u0026ndash;12]. BZW1 plays a key role in the cell cycle by regulating histone H4 gene transcription [13]. Moreover, BZW1 promotes cell proliferation in salivary mucoepidermoid carcinoma and lung adenocarcinoma [14\u0026ndash;16]. BZW1 overexpression is an independent prognostic marker in lung adenocarcinoma [16]. However, the biological function of BZW1 in prostate cancer remains unknown.\u003c/p\u003e \u003cp\u003eHere, the expression of BZW1 and its clinical significance in prostate cancer were investigated. Moreover, the effect of BZW1 expression on cell proliferation and its potential mechanism were explored.\u003c/p\u003e "},{"header":"Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and samples\u003c/h2\u003e \u003cp\u003eA total of 136 prostate cancer and matched adjacent non-cancerous prostate tissues were enrolled in this study. All patients received surgery resection during 2010 to 2018, while no patient treated with chemotherapy or radiotherapy before surgery. Clinicalpathological characters were obtained from hospitalized records. The follow-up time was ranged from 8 to 64 months. This study was supported by the ethics committee of Sun Yat-sen University (No. Z81771573) and performed in accordance with the Declaration of Helsinki. The informed consent was signed by each patient.\u003c/p\u003e \u003c/div\u003e \n\u003ch2\u003eCell Culture And Transfection\u003c/h2\u003e\n \u003cp\u003eHuman prostate cancer cell lines (PC3, Du145 and LnCap) were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were cultured with RPMI-1640 (Gibco) with 10% heat-inactivated fetal bovine serum (FBS, Hyclone) at 37\u0026nbsp;\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cell transfection was performed by Lipofectamine\u0026trade; 2000 (Invitrogen, Thermo Fisher Scientific, Inc, USA) according to the manufacturer's instructions. Then, cells were cultured with 10% heat-inactivated fetal bovine serum (FBS, Hyclone) supplemented with puromycin (0.5\u0026nbsp;\u0026micro;g/mL). BZW1 down-regulation was performed by transferring shRNA lentivirus vectors (Genepharma, Suzhou, China) and BZW1 over-expression was performed by transferring recombinant lentivirus vectors (Genepharma, Suzhou, China). Non-target shRNA (Genepharma, Suzhou, China) lentivirus vectors were used as the negative control (NC).\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eQRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from fresh tissues and cells by using TRIzol Reagent (Life Technologies). According to the manufacture\u0026rsquo;s protocol, the RNA was reversed into cDNA using a PrimeScript RT kit (Takara, Dalian, China). Real-time quantitative PCR was performed by SYBR1 Premix Ex Taq (DDR041A, TaKaRa, Dalian, China). Primers for BZW1 were 5\u0026prime;-ACTGGTGTTCTTCTGGCTAA-3\u0026prime; (Forward) and 5\u0026prime;-GTGCTCATTACACTTGACCA-3\u0026prime; (Reverse). The primer sequence for GAPDH were 5\u0026prime;-CTGAACGGGAAGCTCACTGG-3\u0026prime; (Forward) and 5\u0026prime;-TGAGGTCCACCACCCTGTTG-3\u0026prime; (Reverse). GAPDH was considered as the internal control and each test was repeated three times. The relative expression level of target genes was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \n\u003ch2\u003eWestern Blotting\u003c/h2\u003e\n \u003cp\u003eProteins were extracted by radioimmunoprecipitation assay (RIPA) Lysis buffer (Abcam, Cambridge, MA, USA) and quantified by bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). 40\u0026nbsp;\u0026micro;g of protein per sample was separated onto 10% SDS-PAGE gels and transferred onto polyvinyl difluoride (PVDF) filter membrane (Bio-Rad, Hercules, CA, USA). Then, PVDF membranes were blocked with 5% non-fat dried milk for 2\u0026nbsp;h, and incubated with anti-BZW1 (1: 1000, ABcam Corp, USA), anti-Smad1 (1: 1000, ABcam Corp, USA), anti-p-Smad1 (1: 1000, ABcam Corp, USA), anti-Smad3 (1: 1000, ABcam Corp, USA), anti-p-Smad3 (1: 1000, ABcam Corp, USA), anti-TGF-β1 (1: 2000, ABcam Corp, USA) and anti-GAPDH (1: 2000, ABcam Corp, USA), respectively at 4 ℃ overnight. Finally, membranes were incubated with secondary antibodies (horseradish peroxidase-conjugated anti-rabbit). Signals were developed using an enhanced chemiluminescence reaction kit (Applygen Technologies, Beijing, China).\u003c/p\u003e \n\u003ch2\u003eImmunohistochemistry (ihc)\u003c/h2\u003e\n \u003cp\u003eTissue sections (Two-micrometre-thick) were deparaffinized with xylene and alcohol. Antigen retrieval was completed by sodium citrate buffer (pH 6.0). Then, sections were incubated with rabbit polyclonal BZW1 antibody (Abcam Corp, USA) (diluted 1: 100) overnight at 4\u0026nbsp;\u0026deg;C. Sections were washed with Phosphate-Buffered Saline (PBS) and incubated with biotin-labeled secondary antibodies for 30 minutes at room temperature. Signals were detected by diaminobenzidine tetrahydrochloride (DAB). PBS substituted with primary antibody was regarded as negative controls. The positive controls were performed by lung adenocarcinoma with positive expression of BZW1 [15]. The scores were calculated by the number of positive cells and staining intensity. Percentage of cells was recorded as 1 (\u0026lt;\u0026thinsp;25%), 2 (25%-50%) and 3 (\u0026gt;\u0026thinsp;51%). Staining intensity was recorded as blank (0), weak (1, +), moderate (2, ++) and strong (3, +++). Total score of each section was ranged from 0 to 9. Based on the semiquantitative analysis, BZW1 expression was scored as low-expression (\u0026lt;\u0026thinsp;4) and high-expression (\u0026ge;\u0026thinsp;4) [15].\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell counting kit-8 (CCK-8) analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCCK-8 assay was used to evaluate the cell proliferation. Cells were transferred and seeded in 96-well plates (1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e cell/well) for 5 days. Then, cells were cultured with 10\u0026nbsp;\u0026micro;L of CCK-8 (Dojindo, Kumamoto, Japan) for 3\u0026nbsp;h, and the optical density of each well was examined by a microplate reader (BioTek, Winooski, VT, USA) at 450\u0026nbsp;nm. Each test was repeated in triplicate at the same condition.\u003c/p\u003e \u003cp\u003e \u003cb\u003eColony formation analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTansfected cells (1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e cell/well) were seeded into 6-well plates. After 2 weeks, cells were obtained and fixed with 100% methanol. Then, cells were incubated with 0.1% crystal violet and stained with hemocytometer for counting. Each experiment was repeated in triplicate at the same condition.\u003c/p\u003e \n\u003ch2\u003eTumor Xenograft In Vivo\u003c/h2\u003e\n \u003cp\u003eBalb/c nude mice (4 weeks old) were purchased from Animal Center and kept on specific pathogen-free conditions in accordance with institutional guidelines. To evaluate the effect of BZW1 on tumor growth, 6\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e prostate cancer cells with shRNA lentivirus vectors were subcutaneously injected into the flank regions of legs (5 mice per group). Negative controls (NC) were treated with prostate cancer cells with non-target shRNA lentivirus vectors. After 2 weeks, tumor size and tumor weight were calculated.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data was characterized as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, and analyzed by SPSS software (version 19.0; SPSS, Chicago, IL, USA). The mRNA expression levels of BZW1 between prostate cancer and matched adjacent non-cancerous tissues were analyzed by paired \u003cem\u003et\u003c/em\u003e-test. The correlation between BZW1 expression and clinicalpathological characters was evaluated by Chi-square test (χ\u003csup\u003e2\u003c/sup\u003e). Survival analysis was performed by using the Kaplan-Meier method with log-rank test. Hazard Ratio (HR) was evaluated using the Cox\u0026rsquo;s proportional hazards model. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistical significance.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBZW1 is highly expressed in prostate cancer and correlated with clinicalpathological characters\u003c/h2\u003e \u003cp\u003eTo evaluate the expression of BZW1, the mRNA levels of BZW1 were detected in 136 cases of prostate cancer and matched adjacent non-cancerous prostate tissues by qRT-PCR. Results shown the mRNA levels of BZW1 was significantly greater in prostate cancer compared with those in matched adjacent non-cancerous prostate tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Then, the protein expression levels of BZW1 was detected by IHC. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E, positive staining of BZW1 was mainly located in the cell cytoplasm as brown and yellow. Semiquantitative analysis shown that the high-expression rate of BZW1 in prostate cancer and non-cancerous prostate tissues were 68.4% and 32.4%, and the difference was statistically significant (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBZW1 expression was detected in PCa and non-cancerous prostate tissues by IHC\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTypes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eBZW1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003elow-expression\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehigh-expression\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCa tissues\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43 (31.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e93 (68.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNon-cancerous prostate tissues\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e92 (67.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44 (32.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo investigate the clinical significance of BZW1 in prostate cancer, the correlation between BZW1 expression and clinicalpathological characters was investigated. Results shown BZW1 high-expression was significantly correlated with T stage, lymph node metastasis, PSA and Gleason score (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, no significant correlation was observed in BZW1 expression with age and smoke history (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, Kaplan-Meier analysis revealed that patients with BZW1 high-expression presented unfavorable prognosis than those with BZW1 low-expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, P\u0026thinsp;=\u0026thinsp;0.002). T stage, lymph node metastasis, PSA and Gleason score rather than age and smoke history were significantly correlated with overall survival time (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Moreover, multivariate Cox regression analysis shown that BZW1 expression, T stage, lymph node metastasis, PSA and Gleason score were independent prognostic factors for overall survival in prostate cancer (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBZW1 expression correlated with clinicalpathological characters in PCa\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eClinicalpathological characters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eBZW1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003elow-expression\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ehigh-expression\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.067\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoke history\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.058\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1-T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph node metastasis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePSA ( ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGleason Score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSurvival analysis was performed by Kaplan-Meier method\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean survival time\u003c/p\u003e \u003cp\u003e(Month, 95% CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBZW1 expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow-expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56 (51\u0026ndash;61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh-expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44 (40\u0026ndash;48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48 (43\u0026ndash;53)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.969\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47 (42\u0026ndash;52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoke history\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47 (42\u0026ndash;52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.439\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49 (44\u0026ndash;54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1-T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55 (51\u0026ndash;59)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41 (37\u0026ndash;45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph node metastasis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54 (50\u0026ndash;58)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41 (37\u0026ndash;45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePSA (ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55 (49\u0026ndash;61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.024\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45 (41\u0026ndash;49)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGleason Score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55 (51\u0026ndash;59)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38 (34\u0026ndash;42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrognostic factors were analyzed by Cox\u0026rsquo;s proportional hazards model\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHazard rate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBZW1 (High-expression VS Low-expression)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.394\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.658\u0026ndash;6.947\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (\u0026le;\u0026thinsp;60\u0026nbsp;years VS\u0026thinsp;\u0026gt;\u0026thinsp;60\u0026nbsp;years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.686\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.385\u0026ndash;1.223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.202\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoke history (Positive VS Negative)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.582\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.327\u0026ndash;1.039\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.067\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT stage (T3 VS T1-T2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.878\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.614\u0026ndash;5.132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePSA (\u0026ge;\u0026thinsp;10\u0026nbsp;ng/ml VS\u0026thinsp;\u0026lt;\u0026thinsp;10\u0026nbsp;ng/ml )\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.096\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.056\u0026ndash;4.163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.034\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph node metastasis (Positive VS Negative)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.516\u0026ndash;4.584\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGleason Score (\u0026ge;\u0026thinsp;8 VS\u0026thinsp;\u0026lt;\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.857\u0026ndash;5.635\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBZW1 promotes cell proliferation in prostate cancer by regulating TGF-β1/Smad pathway\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the biological functions of BZW1 in prostate cancer, the influence of BZW1 expression on cell proliferation was investigated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, BZW1 was highly expressed in PC3 cells, but was relatively lower in DU145 and LnCap cells. Thus, BZW1 expression was knocked down in PC3 cells and over-expressed in LnCap cells. QRT-PCR and Western blot analysis shown BZW1 down-regulation and up-regulation were successfully performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Then, cell proliferation was evaluated by CCK-8 and Colony formation assays \u003cem\u003ein vitro\u003c/em\u003e. CCK-8 assay showed that BZW1 knockdown significantly inhibited the proliferation in PC3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), while BZW1 over-expression significantly promoted the proliferation in LnCap cells. The colony number in cells with BZW1 down-regulation was significantly decreased, while was significantly increased in cells with BZW1 over-expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To further investigate the effect of BZW1 expression on cell proliferation, xenografts \u003cem\u003ein vivo\u003c/em\u003e was performed. Results showed that the size and weight of tumor with BZW1 knockdown were significantly decreased compared with those in NC groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore the potential mechanism of BZW1, TGF-β1/Smad pathway was investigated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, the expression of TGF-β1 was significantly decreased with the knockdown of BZW1, while was significantly up-regulated by BZW1 over-expression. The protein expression levels of Smad1 and Smad3 were unchanged regardless of whether the BZW1 expression interference, while BZW1 down-regulation resulted in the decrease of p-Smad1 and p-Smad3.\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eBZW1, as one of transcription factors, encode a N-terminal bZIP domain and a C-terminal nucleotide binding domain [11\u0026ndash;12]. The proteins and transcription factors of bZIP domain play crucial roles in cancer development [17\u0026ndash;18]. Recently, BZW1 over-expression was observed in mucoepidermoid carcinoma [14] and lung adenocarcinoma [16]. However, the role of BZW1 in prostate cancer remains unknown.\u003c/p\u003e \u003cp\u003eHere, the expression and clinical significance of BZW1 was investigated in prostate cancer. Results shown that the mRNA and protein levels of BZW1 in prostate cancer were significantly higher compared with those in non-cancerous tissues. These data indicated that BZW1 overexpression was associated with the formation of prostate cancer, which was consistent with the observations in mucoepidermoid carcinoma [14] and lung adenocarcinoma [16]. BZW1 over-expression might be helpful for the diagnosis of prostate cancer. In addition, BZW1 over-expression significantly correlated with T stage, lymph node metastasis, PSA and Gleason score, suggesting that BZW1 over-expression was connected with the progression of PCa. Moreover, survival analysis shown that BZW1 as well as T stage, lymph node metastasis, PSA and Gleason score were associated with patients\u0026rsquo; survival, which could be as independent prognostic factors in PCa. Moreover, Chiou et al [16] reported BZW1 over-expression was an independent poor prognosis marker in lung adenocarcinoma.\u003c/p\u003e \u003cp\u003eSubsequently, the influence of BZW1 expression on cell proliferation and its potential mechanism were explored. Results shown BZW1 down-regulation significantly suppressed cell proliferation, while BZW1 over-expression significantly promoted cell proliferation in \u003cem\u003evitro\u003c/em\u003e. Xenografts assays further validated that BZW1 knockdown significantly inhibited the proliferation of prostate cancer \u003cem\u003ein vivo\u003c/em\u003e. To explore the potential mechanism of BZW1, TGF-β1/Smad pathway was investigated. It is well known that TGF-β/Smad pathway is involved in cell proliferation [19\u0026ndash;21]. Results shown that BZW1 over-expression activated the TGF-β1/Smad pathway. Thus, these data indicated that BZW1 over-expression promoted cell proliferation, which was connected with the TGF-β1/Smad pathway.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eThese data indicate that BZW1 is highly expressed in prostate cancer and correlates with tumor progression and prognosis. Moreover, BZW1 overexpression promotes cell proliferation by regulating TGF-β1/Smad pathway.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDeclarations\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNot applicable.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKL conceived and designed the study. TCL and ZLS performed the analysis. JYW and CTX coordinated the statistical analyses of results. KL and TCL wrote the manuscript. All authors read and approved the manuscript. All authors consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is supported by the Pearl River S\u0026amp;T Nova Program of Guangzhou (No. 201710010039), Basic Service Charge Young Teachers Cultivation Project of Sun Yat-sen University (17ykpy48) and National Natural Science Foundation (No. 81771573). The design of the study, collection of samples, analysis of results, interpretation of data, and writing of the manuscript was done by the authors (for details on authors\u0026rsquo; contribution see below).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was reviewed and approved by the internal review boards (IRB) of the involved institutions: Sun Yat-sen University. (No. Z81771573).\u003c/p\u003e\n\u003cp\u003eAll eligible subjects signed an informed consent to participate in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll author declare they have no financial disclosures and conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e1. Li Z, Wei D, Zhu C, Zhang Q. Effect of a patient education and rehabilitation program on anxiety, depression and quality of life in muscle invasive\u0026ensp;Prostate cancer\u0026ensp;patients treated with adjuvant chemotherapy. Medicine (Baltimore). 2019, 98(44): e17437.\u003c/p\u003e\n\u003cp\u003e2. Zhao F, Yu X, Xu M, Ye S, Zang S, Zhong W, Ren G, Chen X, Yan S. Mucinous\u0026ensp;Prostate\u0026ensp;Cancer\u0026ensp;Shows\u0026ensp;Similar\u0026ensp;Prognosis\u0026ensp;to Typical\u0026ensp;Prostate\u0026ensp;Acinar Carcinoma: A Large Population-Based and Propensity Score-Matched Study. Front Oncol. 2020, 9:1467.\u003c/p\u003e\n\u003cp\u003e3. Kimura T, Egawa S. Epidemiology of prostate cancer in Asian countries. Int J Urol, 2018, 25(6): 524\u0026ndash;531.\u003c/p\u003e\n\u003cp\u003e4. Sakamoto S. Editorial Comment to Epidemiology of prostate cancer in Asian countries. Int J Urol, 2018, 25(6): 531\u0026ndash;532.\u003c/p\u003e\n\u003cp\u003e5. Saika T, Miura N, Fukumoto T, Yanagihara Y, Miyauchi Y, Kikugawa T. Role of robot-assisted radical prostatectomy in locally advanced prostate cancer. Int. J. Urol. 2018, 25: 30\u0026ndash;35.\u003c/p\u003e\n\u003cp\u003e6. Center MM, Jemal A, Lortet-Tieulent J, Ward E, Ferlay J, Brawley O, Bray F. International variation in prostate cancer incidence and mortality rates. Eur. 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Kodama H, Hatakeyama S, Narita S, Takahashi M, Sakurai T, Kawamura S, Hoshi S, Ishida M, Kawaguchi T, Ishidoya S, Shimoda J, Narita T, Sato H, Mitsuzuka K,Tochigi T, Tsuchiya N, Arai Y, Habuchi T, Ohyama C. Clinical Characterization of Low Prostate-specific Antigen on Prognosis in Patients With Metastatic Castration-naive Prostate Cancer. Clin Genitourin Cancer. 2019, 17(6): e1091-e1098.\u003c/p\u003e\n\u003cp\u003e11. Machado RD, Pauciulo MW, Fretwell N, Veal C, Thomson JR, Vilarino Guell C, Aldred M, Brannon CA, Trembath RC, Nichols WC. A physical and transcript map based upon refinement of the critical interval for PPH1, a gene for familial primary pulmonary hypertension. The International PPH Consortium. Genomics. 2000, 68(2): 220\u0026ndash;228.\u003c/p\u003e\n\u003cp\u003e12. Nomura N, Nagase T, Miyajima N, Sazuka T, Tanaka A, Sato S, Seki N, Kawarabayasi Y, Ishikawa K, Tabata S. Prediction of the coding sequences of unidentified human genes. II. 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J Cell Sci. 2013, 126(Pt 8): 1858\u0026ndash;1867.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"BZW1, prognosis, Prostate cancer, survival, biomarker","lastPublishedDoi":"10.21203/rs.3.rs-45376/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-45376/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e Recently, basic leucine zipper and the W2 domain-containing protein 1 (BZW1) is reported to be implicated in tumor progression. However, the role of BZW1 in prostate cancer remains unknown. This study is aimed to investigate the expression of BZW1 and its influence on cell proliferation in prostate cancer. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e The expression levels of BZW1 were measured in 136 cases of prostate cancer and matched adjacent non-cancerous prostate tissues by quantificational real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). Then, the effect of BZW1 on cell proliferation was further explored. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eQRT-PCR analysis shown that the mRNA levels of BZW1 in prostate cancer were significantly greater compared with those in matched adjacent non-cancerous prostate tissues (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). IHC results shown the high-expression rate of BZW1 in prostate cancer and matched adjacent non-cancerous prostate tissues were 68.4% and 32.4%, and the difference was statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). BZW1 high-expression significantly correlated with T stage, lymph node metastasis, prostate specific antigen (PSA) and Gleason score (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). Patients with BZW1 high-expression presented unfavorable prognosis compared with those with BZW1 low-expression (\u003cem\u003eP\u003c/em\u003e=0.002). In addition, CCK-8 and Colony formation assays revealed that BZW1 over-expression significantly promoted cell proliferation in \u003cem\u003evitro\u003c/em\u003e. Tumor xenograft shown BZW1 knockdown significantly inhibited tumor growth in vivo. Moreover, BZW1 overexpression activated the TGF-β1/Smad1/Smad3 pathway. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e BZW1 over-expression predicts poorer prognosis and promotes cell proliferation in prostate cancer by regulating TGF-β1/Smad pathway.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"BZW1 Promotes Cell Proliferation in Prostate Cancer by Regulating TGF-β1/Smad Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-08-07 14:49:08","doi":"10.21203/rs.3.rs-45376/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6f0dfa9a-3b10-411a-bcf5-6303a08021bd","owner":[],"postedDate":"August 7th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":256870,"name":"Cancer Biology"},{"id":256871,"name":"Oncology"}],"tags":[],"updatedAt":"2020-09-20T17:08:36+00:00","versionOfRecord":[],"versionCreatedAt":"2020-08-07 14:49:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-45376","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-45376","identity":"rs-45376","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}