Supplementary Material for: The Search for Secreted Proteins in Prostate Cancer by the <b><i>Escherichia coli</i></b> Ampicillin Secretion Trap: Expression of NBL1 Is Highly Restricted to the Prostate and Is Related to Cancer Progression

<b><i>Aims:</i></b> Genes expressed only in cancer tissue or specific organs will be useful molecular markers. To identify genes that encode secreted proteins present in prostate cancer (PCa), we generated <i>Escherichia coli</i> ampicillin secretion trap (CAST) libraries from PCa and normal prostate (NP). <b><i>Methods and Results:</i></b> We identified 15 candidate genes that encode secreted proteins present in PCa and NP. Quantitative RT-PCR analysis revealed that <i>MSMB</i>, <i>NBL1</i> and <i>AZGP1</i> were expressed with much higher specificity in PCa and NP than in 14 other kinds of normal tissue. We focused on <i>NBL1</i>, which was originally identified as a putative tumor suppressor gene. Western blot analysis revealed that NBL1 protein was highly expressed in both cell lysate and culture media of the DU145 PCa cell line. Immunohistochemical analysis showed that NBL1 expression was highly detected in and restricted to NP and PCa and was significantly down-regulated in PCa. NBL1 expression was significantly reduced according to the tumor stage, Gleason grade and preoperative prostate-specific antigen (PSA) value. <b><i>Conclusion:</i></b> NBL1 is a secreted protein that is highly restricted to the prostate. Underexpression of NBL1 correlated with PCa progression. NBL1 might be a candidate tumor marker for PCa in addition to PSA.<b><i>研究目标：</i></b> 仅在癌症组织或特定器官中表达的基因，可作为极具应用价值的分子标记物。为识别编码存在于前列腺癌（prostate cancer, PCa）中的分泌蛋白的基因，我们从前列腺癌与正常前列腺（normal prostate, NP）样本中构建了大肠杆菌（<i>Escherichia coli</i>）氨苄青霉素分泌陷阱（ampicillin secretion trap, CAST）文库。<b><i>方法与结果：</i></b> 本研究共鉴定出15个可编码前列腺癌与正常前列腺中分泌蛋白的候选基因。定量逆转录聚合酶链反应（quantitative RT-PCR）分析结果显示，MSMB、NBL1与AZGP1在前列腺癌和正常前列腺中的表达特异性远高于其余14种正常组织。我们将研究重点聚焦于NBL1——该基因最初被鉴定为潜在的肿瘤抑制基因。蛋白质免疫印迹（Western blot）分析表明，NBL1蛋白在DU145前列腺癌细胞系的细胞裂解液与细胞培养基中均呈高表达。免疫组化（immunohistochemical）分析结果显示，NBL1的表达仅在正常前列腺与前列腺癌组织中可被高效检出，且在前列腺癌组织中其表达水平显著下调。NBL1的表达水平随肿瘤分期、格利森分级（Gleason grade）以及术前前列腺特异性抗原（prostate-specific antigen, PSA）值的升高而显著降低。<b><i>结论：</i></b> NBL1是一种高度局限于前列腺组织的分泌蛋白。NBL1的低表达与前列腺癌的进展密切相关。除前列腺特异性抗原（PSA）外，NBL1或可成为前列腺癌的候选肿瘤标志物。