iTRAQ-Multidimensional Liquid Chromatography and Tandem Mass Spectrometry-Based Identification of Potential Biomarkers of Oral Epithelial Dysplasia and Novel Networks between Inflammation and Premalignancy
iTRAQ-Multidimensional Liquid Chromatography and Tandem Mass Spectrometry-Based Identification of Potential Biomarkers of Oral Epithelial Dysplasia and Novel Networks between Inflammation and Premalignancy
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DOI：
10.1021/pr800501j
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发表时间：
2009-01-01
影响因子：
4.4
通讯作者：
Siu, K. W. Michael
中科院分区：
文献类型：
--
作者：
Ralhan, Ranju;DeSouza, Leroi V.;Siu, K. W. Michael
口腔粘膜长期暴露于烟草中的致癌物与炎症和口腔癌前病变（OPL）的发展有关，具有进展为癌症的高风险;目前尚无临床方法来识别高风险病变。我们假设，使用蛋白质组学方法鉴定OPL中与正常口腔组织相关的差异表达蛋白质将揭示多个细胞通路的变化，并有助于生物标志物的发现。同位素质量标签（iTRAQ）标记的口腔发育不良和正常组织与合并的正常对照进行了比较，通过在线液相色谱和串联质谱。通过免疫组织化学、免疫印迹和RT-PCR在一组独立的样品中进行生物标志物的验证。我们确定了459个非冗余蛋白质的OPL，包括结构蛋白，信号成分，酶，受体，转录因子和伴侣。通过iTRAQ分析鉴定并经免疫组化验证的三种表现最好的生物标志物--分层（SFN）、YWHAZ和hnRNPK--在区分发育不良和正常组织中分别达到了0.83、0.91的灵敏度、0.74、0.95的特异性和0.87和0.96的预测值，从而证实了它们作为潜在OPL生物标志物的实用性。通路分析揭示了所有三种生物标志物之间的直接相互作用，以及它们参与炎症、信号传导、增殖、基因表达调控和癌症的两个主要网络。总之，我们确定OPL蛋白质组的工作揭示了连接炎症和上皮发育异常及其关键调控蛋白的新网络，可作为早期干预的新化学预防/治疗靶点。此外，我们确定并验证了一组OPL生物标志物，这些生物标志物有望用于最终临床应用的大规模验证。
Chronic exposure of the oral mucosa to carcinogens in tobacco is linked to inflammation and development of oral premalignant lesions (OPLs) with high risk of progression to cancer; there is currently no clinical methodology to identify high-risk lesions. We hypothesized that identification of differentially expressed proteins in OPLs in relation to normal oral tissues using proteomic approach will reveal changes in multiple cellular pathways and aid in biomarker discovery. Isobaric mass tags (iTRAQ)-labeled oral dysplasias and normal tissues were compared against pooled normal control by online liquid chromatography and tandem mass spectrometry. Verification of biomarkers was carried out in an independent set of samples by immunohistochemistry, immunoblotting, and RT-PCR. We identified 459 nonredundant proteins in OPLs, including structural proteins, signaling components, enzymes, receptors, transcription factors, and chaperones. A panel of three best-performing biomarkers identified by iTRAQ analysis and verified by immunohistochemistry-stratifin (SFN), YWHAZ, and hnRNPK-achieved a sensitivity of 0.83,0.91, specificity of 0.74, 0.95, and predictive value of 0.87 and 0.96, respectively, in discriminating dysplasias from normal tissues, thereby confirming their utility as potential OPL biomarkers. Pathway analysis revealed direct interactions between all the three biomarkers and their involvement in two major networks involved in inflammation, signaling, proliferation, regulation of gene expression, and cancer. In conclusion, our work on determining the OPL proteome unraveled novel networks linking inflammation and development of epithelial dysplasia and their key regulatory proteins may serve as novel chemopreventive/therapeutic targets for early intervention. Additionally, we identified and verified a panel of OPL biomarkers that hold promise for large-scale validation for ultimate clinical use.