Molecular characterization of 4-NQO induced F344 rat tongue carcinogenesis: alteration of multiple gene expression and hypomethylation of PTGS2 proximal promoter

The 4-NQO induced rat tongue carcinogenesis model presenting considerable histologic and molecular similarity to oral cancers commonly seen in humans is very useful for investigating oral carcinogenesis. In order to understand the molecular basis of oral carcinogenesis and identify gene biomarkers and potential chemopreventive targets for future studies, we characterized the molecular changes in oral squamous cell carcinoma (SCC) samples generated from this model system using F344 rats by performing gene expression microarray and methylation analysis of selected genes. Microarray studies identified 1735 genes to be upregulated by at least 2 fold (p<0.05, n=11) and 1803 genes to be downregulated by at least 50% (p<0.05, n=11) in SCC in comparison to the adjacent normal tissues and 28 KEGG pathways to be altered in SCC (p<0.01). Among the altered genes, keratins and keratin associated proteins were found to be differentially regulated and the keratin profile appeared to an important biomarker for oral SCC. PTGS2 and PTGS2 relevant genes, which are potential targets of chemoprevention and therapy, were also found to be upregulated in SCC and confirmed by qRT-PCR. The upregulation of PTGS2 appeared to correlate with hypomethylation of its proximal promoter. Methylation analysis of other selected genes showed that the first exon of APC2 was methylated in normal tissues, and the methylation level increased moderately in SCC samples (p<0.01, n=8). These results demonstrate that 4-NQO induced tongue carcinogenesis in F344 rats is accompanied by alteration of multiple gene expression, hypomethylation of PTGS2 and increased methylation of APC2.由4-NQO诱导的大鼠舌癌变模型，其组织学与分子特征与人类常见口腔癌具有高度相似性，是研究口腔癌变机制的理想工具。为阐明口腔癌变的分子基础，并筛选可用于后续研究的基因生物标志物与潜在化学预防靶点，本研究以F344大鼠构建的该模型系统所产生的口腔鳞状细胞癌（oral squamous cell carcinoma, SCC）样本为研究对象，通过基因表达微阵列分析与候选基因甲基化检测，对其分子变化特征进行了系统表征。微阵列分析结果显示，与癌旁正常组织相比，口腔鳞状细胞癌样本中共有1735个基因表达上调至少2倍（p<0.05，n=11）、1803个基因表达下调至少50%（p<0.05，n=11），同时存在28条KEGG通路发生显著改变（p<0.01）。在发生表达改变的基因中，角蛋白与角蛋白关联蛋白均呈现显著差异表达，其表达谱或可作为口腔鳞状细胞癌的重要生物标志物。作为潜在的化学预防与治疗靶点，前列腺素内过氧化物合酶2（PTGS2）及其相关基因在口腔鳞状细胞癌中亦呈现上调表达，该结果经实时定量逆转录聚合酶链反应（quantitative real-time PCR, qRT-PCR）验证。PTGS2的上调表达与其近端启动子区域的低甲基化状态显著相关。对其余候选基因的甲基化检测结果显示，正常组织中APC2基因的第一外显子存在甲基化修饰，而在口腔鳞状细胞癌样本中其甲基化水平呈中度升高（p<0.01，n=8）。上述实验结果表明，F344大鼠体内由4-NQO诱导的舌癌变过程，伴随多基因表达异常、PTGS2基因低甲基化以及APC2基因甲基化水平升高。