Solmi Rossella, De Sanctis Paola, Zucchini Cinzia, Ugolini Giampaolo, Rosati Giancarlo, Del Governatore Marco, Coppola Domenico, Yeatman Thymothy J, Lenzi Luca, Caira Antonello, Zanotti Simone, Taffurelli Mario, Carinci Paolo, Valvassori Luisa, Strippoli Pierluigi
Institute of Histology and General Embriology, University of Bologna, Fondazione CARISBO Centre for Research into Molecular Genetics, I-40126 Bologna, Italy.
Int J Oncol. 2004 Oct;25(4):1049-56.
Research has widely supported the efficacy of screening for colorectal cancer in reducing mortality. A blood-based assay potentially represents a more accessible early detection tool for the identification of solid tumor cells originating from a primary tumor site in the body. We demonstrate a relatively easy and highly reproducible technique for the detection of mRNA expression of genes as markers of malignancy in blood samples of patients with colon cancer. The present study aims to identify a set of specific mRNAs expressed in epithelial cells but not in blood cells, which may be useful as markers for early detection of circulating colon cancer cells by a simple, qualitative RT-PCR assay following semi-automated RNA extraction from peripheral blood samples. Our approach includes a systematic search for candidate markers using digital differential display, search on UniGene colon EST libraries and analysis of published data on colon cancer gene expression. A final list included the following genes: bone morphogenetic protein 4 (BMP4), cyclin D (CycD), family with sequence similarity 3, member D (FAM3D), gastrin (GAS), glycoprotein A33 transmembrane (GPA33), glutathione peroxidase 2 gastrointestinal (GPX2), galactoside-binding, soluble, 4 (galectin 4) (LGALS4), non-SMC, structural maintenance of chromosomes, element 1 protein (NSE1), tumor-associated calcium signal transducer 1 (TACSTD1), telomerase reverse transcriptase (hTERT), trefoil factor 3 intestinal (TFF3), transmembrane 4 superfamily member 3 (TM4SF3), UDP glycosyltransferase 1 family, polypeptide A9 (UGT1A9), villin 1 (VIL1), and the novel gene FLJ20127. The mRNA expression of these genes was evaluated in a pool of 16 samples from subjects diagnosed with colon cancer and from 16 normal-controls. We observed expression in 13 of the 15 investigated genes from the blood samples of the vast majority of patients considered, but also in a certain percentage of the controls (from 14.3 to 100%). This finding confirms that the extreme sensitivity of RT-PCR is able to detect minimal amounts of mRNA expressed in a non tissue-specific manner ('illegitimate transcription'). On the contrary, NSE1 and GAS mRNAs were not detected either in patient or in control blood samples; however, they were abundantly expressed in normal and cancerous colon mucosa, encouraging further search for useful markers able to detect epithelial cells in peripheral blood.
研究广泛支持结直肠癌筛查在降低死亡率方面的功效。一种基于血液的检测方法可能代表了一种更容易获得的早期检测工具，用于识别源自身体原发肿瘤部位的实体瘤细胞。我们展示了一种相对简单且高度可重复的技术，用于检测结肠癌患者血液样本中作为恶性肿瘤标志物的基因的mRNA表达。本研究旨在鉴定一组在上皮细胞中表达但在血细胞中不表达的特定mRNA，这可能有助于通过从外周血样本中进行半自动RNA提取后进行的简单定性逆转录聚合酶链反应（RT-PCR）检测来早期检测循环中的结肠癌细胞。我们的方法包括使用数字差异显示系统搜索候选标志物、在UniGene结肠EST文库中搜索以及分析关于结肠癌基因表达的已发表数据。最终列表包括以下基因：骨形态发生蛋白4（BMP4）、细胞周期蛋白D（CycD）、序列相似性家族3成员D（FAM3D）、胃泌素（GAS）、糖蛋白A33跨膜蛋白（GPA33）、胃肠道谷胱甘肽过氧化物酶2（GPX2）、半乳糖苷结合可溶性蛋白4（galectin 4）（LGALS4）、非SMC染色体结构维持元件1蛋白（NSE1）、肿瘤相关钙信号转导蛋白1（TACSTD1）、端粒酶逆转录酶（hTERT）、肠三叶因子3（TFF3）、跨膜4超家族成员3（TM4SF3）、UDP糖基转移酶1家族多肽A9（UGT1A9）、绒毛蛋白1（VIL1）以及新基因FLJ20127。在来自被诊断患有结肠癌的受试者和16名正常对照的16个样本池中评估了这些基因的mRNA表达。我们观察到在所考虑的绝大多数患者的血液样本中，15个研究基因中的13个有表达，但在一定比例的对照中也有表达（从14.3%到100%）。这一发现证实了RT-PCR的极高灵敏度能够检测以非组织特异性方式表达的极少量mRNA（“非法转录”）。相反，在患者或对照血液样本中均未检测到NSE1和GAS的mRNA；然而，它们在正常和癌性结肠黏膜中大量表达，这鼓励进一步寻找能够检测外周血中上皮细胞的有用标志物。
