Department of Proteomics, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden.
Proteomics Clin Appl. 2011 Dec;5(11-12):624-35. doi: 10.1002/prca.201100020.
In this study, we investigated the prognostic impact of human RBM3 expression in colorectal cancer using tissue microarray-based immunohistochemical analysis.
One polyclonal antibody and four monoclonal anti-RBM3 antibodies were generated and epitope mapped using two different methods. Bacterial display revealed five distinct epitopes for the polyclonal antibody, while the four mouse monoclonal antibodies were found to bind to three of the five epitopes. A peptide suspension bead array assay confirmed the five epitopes of the polyclonal antibody, while only one of the monoclonal antibodies could be mapped using this approach. Antibody specificity was confirmed by Western blotting and immunohistochemistry, including siRNA-mediated knock-down. Two of the antibodies (polyclonal and monoclonal) were subsequently used to analyze RBM3 expression in tumor samples from two independent colorectal cancer cohorts, one consecutive cohort (n=270) and one prospectively collected cohort of patients with cancer of the sigmoid colon (n=305). RBM3-expression was detected, with high correlation between both antibodies (R=0.81, p<0.001).
In both cohorts, tumors with high nuclear RBM3 staining had significantly prolonged the overall survival. This was also confirmed in multivariate analysis, adjusted for established prognostic factors.
These data demonstrate that high tumor-specific nuclear expression of RBM3 is an independent predictor of good prognosis in colorectal cancer.
本研究通过基于组织微阵列的免疫组织化学分析，探讨人类 RBM3 表达在结直肠癌中的预后影响。
生成了一种多克隆抗体和四种抗 RBM3 单克隆抗体，并使用两种不同的方法进行了表位作图。细菌展示揭示了多克隆抗体的五个独特表位，而四种鼠单克隆抗体被发现与其中的三个表位结合。肽悬浮珠阵列测定法证实了多克隆抗体的五个表位，而只有一种单克隆抗体可以通过这种方法进行定位。通过 Western blot 和免疫组织化学（包括 siRNA 介导的敲低）证实了抗体的特异性。随后，使用两种抗体（多克隆和单克隆）分析了来自两个独立结直肠癌队列的肿瘤样本中的 RBM3 表达，一个是连续队列（n=270），另一个是前瞻性收集的乙状结肠癌患者队列（n=305）。检测到 RBM3 表达，两种抗体之间具有高度相关性（R=0.81，p<0.001）。
在两个队列中，核 RBM3 染色高的肿瘤具有显著延长的总生存期。这在多变量分析中也得到了证实，调整了既定的预后因素。
这些数据表明，结直肠癌中肿瘤特异性核表达 RBM3 高是预后良好的独立预测因子。
