Sun Stella, Yi Xin, Poon Ronnie Tp, Yeung Chun, Day Philip J R, Luk John M
Department of Surgery, LKS Faculty of Medicine, Jockey Club Clinical Research Centre, The University of Hong Kong, Pokfulam, Hong Kong.
BMC Cancer. 2009 Sep 2;9:309. doi: 10.1186/1471-2407-9-309.
During the last decade, investigations have focused on revealing genes or proteins that are involved in HCC carcinogenesis using either genetic or proteomic techniques. However, these studies are overshadowed by a lack of good internal reference standards. The need to identify "housekeeping" markers, whose expression is stable in various experimental and clinical conditions, is therefore of the utmost clinical relevance in quantitative studies. This is the first study employed 2-DE analysis to screen for potential reference markers and aims to correlate the abundance of these proteins with their level of transcript expression.
A Chinese cohort of 224 liver tissues samples (105 cancerous, 103 non-tumourous cirrhotic, and 16 normal) was profiled using 2-DE analysis. Expression of the potential reference markers was confirmed by western blot, immunohistochemistry and real-time quantitative PCR. geNorm algorithm was employed for gene stability measure of the identified reference markers.
The expression levels of three protein markers beta-actin (ACTB), heat shock protein 60 (HSP60), and protein disulphide isomerase (PDI) were found to be stable using p-values (p > 0.99) as a ranking tool in all 224 human liver tissues examined by 2-DE analysis. Of high importance, ACTB and HSP 60 were successfully validated at both protein and mRNA levels in human hepatic tissues by western blot, immunohistochemistry and real-time quantitative PCR. In addition, no significant correlation of these markers with any clinicopathological features of HCC and cirrhosis was found. Gene stability measure of these two markers with other conventionally applied housekeeping genes was assessed by the geNorm algorithm, which ranked ACTB and HSP60 as the most stable genes among this cohort of clinical samples.
Our findings identified 2 reference markers that exhibited stable expression across human liver tissues with different conditions thus should be regarded as reliable reference moieties for normalisation of gene and protein expression in clinical research employing human hepatic tissues.
在过去十年中，研究主要集中于使用基因或蛋白质组学技术揭示参与肝癌发生的基因或蛋白质。然而，这些研究因缺乏良好的内部参考标准而黯然失色。因此，识别在各种实验和临床条件下表达稳定的“管家”标志物对于定量研究具有至关重要的临床意义。这是第一项采用二维电泳分析筛选潜在参考标志物并旨在将这些蛋白质的丰度与其转录表达水平相关联的研究。
使用二维电泳分析对224例肝组织样本（105例癌组织、103例非肿瘤性肝硬化组织和16例正常组织）的中国队列进行分析。通过蛋白质印迹、免疫组织化学和实时定量PCR确认潜在参考标志物的表达。采用geNorm算法对鉴定出的参考标志物进行基因稳定性测定。
在通过二维电泳分析检测的所有224例人类肝组织中，使用p值（p>0.99）作为排名工具，发现三种蛋白质标志物β-肌动蛋白（ACTB）、热休克蛋白60（HSP60）和蛋白质二硫键异构酶（PDI）的表达水平稳定。至关重要的是，通过蛋白质印迹、免疫组织化学和实时定量PCR在人类肝组织的蛋白质和mRNA水平上成功验证了ACTB和HSP 60。此外，未发现这些标志物与肝癌和肝硬化的任何临床病理特征有显著相关性。通过geNorm算法评估了这两种标志物与其他常规应用的管家基因的基因稳定性，该算法将ACTB和HSP60列为该临床样本队列中最稳定的基因。
我们的研究结果确定了2种参考标志物，它们在不同条件的人类肝组织中表现出稳定的表达，因此应被视为在使用人类肝组织的临床研究中用于基因和蛋白质表达标准化的可靠参考部分。
