Clinical significance of UNC5B Expression in Colorectal Cancer. Homo sapiens

Purpose and Experimental Design: The purpose of this study is to find a methylation-related gene that could become a biomarker or therapeutic target in colorectal carcinoma (CRC). We screened candidate genes suspected to be silenced by DNA methylation using oligonucleotide microarray analysis. To investigate the clinical significance of one candidate gene (UNC5B), we analyzed the correlation between mRNA expression and clinicopathological features using clinical tissue samples. Finally, methylation specific PCR analysis was performed to reveal whether the promoter region was methylated in CRC cell lines. Results: We found 75 candidate genes that were potentially suppressed by DNA methylation in CRC. We focused on UNC5B, a possible tumor suppressor gene and regulator of apoptosis known to be inactivated in CRC. The mRNA expression analysis using tissue samples revealed that UNC5B mRNA was down-expressed in about 20% of CRC patients, and the patients with low-UNC5B-expression tumors showed a significantly higher recurrence rate after curative surgery. According to the univariate and multivariate analysis, low UNC5B expression was an independent risk factor for postoperative recurrence in stage I, II, and III CRC patients. Furthermore, patients with low expression of UNC5B in tumors had significantly poorer prognosis than those with high expression of UNC5B. Although UNC5B mRNA expression was restored by the demethylation treatment in CRC cell lines, the promoter region of UNC5B was not methylated. Conclusion: UNC5B is a potential biomarker for the selection of patients with high risk of postoperative recurrence and worse prognosis in CRC. Overall design: Gene expression profiles for 17 pairs of cancer and non-cancerous tissues from colorectal cancer patients were measured by Affymetrix HG-U133 Plus 2.0 arrays. HT29 and HCT15 cells were also used to investigate genes upregulated after 5-aza-2'-deoxycytidine treatment. Normalization was performed by robust multi-array average (RMA) method under R 2.6.2 statistical software with affy package from BioConductor. The normalization procedure was separately performed for each data set of clinical samples, HT29 cells, and HCT15 cells. The normalized gene expression levels were presented as log2-transformed values by RMA.

研究目的与实验设计：本研究旨在探寻结直肠癌（colorectal carcinoma, CRC）中可作为生物标志物或治疗靶点的甲基化相关基因。本研究通过寡核苷酸微阵列分析（oligonucleotide microarray analysis）筛选疑似被DNA甲基化沉默的候选基因；为探究候选基因UNC5B的临床意义，利用临床组织样本分析其mRNA表达与临床病理特征的相关性；最终通过甲基化特异性PCR（methylation specific PCR）分析，明确CRC细胞系中UNC5B启动子区域是否发生甲基化。

研究结果：本研究共筛选出75个在CRC中可能被DNA甲基化抑制的候选基因，并重点关注UNC5B——这是一种已知在CRC中失活的潜在抑癌基因及凋亡调节因子。对临床组织样本的mRNA表达分析显示，约20%的CRC患者肿瘤组织中UNC5B mRNA表达下调；且UNC5B低表达的肿瘤患者在接受根治性手术后，复发率显著更高。单因素分析（univariate analysis）及多因素分析（multivariate analysis）结果表明，UNC5B低表达是I、II、III期CRC患者术后复发的独立危险因素。此外，肿瘤组织中UNC5B低表达的患者，其预后显著差于UNC5B高表达患者。尽管在CRC细胞系中经去甲基化处理（demethylation treatment）可恢复UNC5B的mRNA表达，但UNC5B启动子区域并未发生甲基化。

研究结论：UNC5B可作为筛选CRC术后复发高风险及预后不良患者的潜在生物标志物。

整体实验设计：本研究采用Affymetrix HG-U133 Plus 2.0芯片，检测17对结直肠癌患者的癌组织与配对癌旁正常组织的基因表达谱；同时利用HT29及HCT15细胞系，探究5-氮杂-2'-脱氧胞苷（5-aza-2'-deoxycytidine）处理后上调的基因。本研究使用R 2.6.2统计软件，结合BioConductor的affy包（affy package from BioConductor），通过稳健多数组平均（robust multi-array average, RMA）方法完成数据标准化，且临床样本、HT29细胞及HCT15细胞的数据集分别独立执行标准化流程。经RMA处理后的标准化基因表达水平以log2转换值（log2-transformed values）形式呈现。