基于电化学发光检测技术的乳腺癌生物标记物EZH2表达定量分析

摘要

Zeste同源染色体2增强子基因(EZH2)在人类乳腺癌中过度表达,它可以被视为一个检测肿瘤的发展和转移的生物标记物.传统技术检测或定量特异性基因表达存在一些缺点,因此,本研究拟开发电致化学发光(ECL)技术来检测和量化EZH2 mRNA的表达量.在本研究中,用生物素和三(2,2 - 联吡啶)钌(II)(TBR)分别标记在PCR引物的5′ 末端上,用作扩增靶基因,扩增产物用ECL系统进行检测.我们用癌细胞作为模型分析了该方法的有效性和灵敏度,并且将其应用于25例乳腺癌的临床样本中EZH2基因表达量的检测.检测结果表明,EZH2基因在肿瘤细胞系中过量表达,而在正常血细胞中则低表达.最重要的是,在25例临床乳腺癌样品中发现10例样品(40%)的EZH2 mRNA过度表达.此方法提供了一种新的工具来评估EZH2基因在乳腺癌中的表达水平,且有可能成为一种快速、简便和灵敏的乳腺癌检测和诊断方法.%The enhancer of zeste homolog 2 ( EZH2 ) is overexpressed in human breast cancer. It can be a biomarker for detection of cancer progression and metastasis. The traditional techniques described to detect or quantify specific gene expression are all have some disadvantages. Thus, the electrochemiluminescence ( ECL ) assay was developed to detect and quantify the amount of EZH2 mRNA expression. In this report, the PCR primers labeled with biotin and tris (2,2-bipyridine ) ruthenium ( Ⅱ ) ( TBR ) on the 5' terminal, respectively, were employed to amplify target gene. The amplification product was detected by ECL system. The method was applied to detect possible EZH2 overexpression in 25 breast cancer clinical samples. Analytical efficacy and sensitivity were demonstrated in model system experiment using cancer cells. Results showed that the EZH2 overexpression could be detected in cancer cells whereas it could not be detected in normal blood cells. Most importantly, the EZH2 mRNA overexpression was found in 10 out of 25 ( 40% ) breast cancer clinical samples. This assay offers a new tool to evaluate EZH2 expression in breast cancer and may potentially become a rapid, simple and sensitive approach for breast cancer detection and diagnostics.