Sun Yuan-Wan, Chen Kun-Ming, Imamura Kawasawa Yuka, Salzberg Anna C, Cooper Timothy K, Caruso Carla, Aliaga Cesar, Zhu Junjia, Gowda Krishne, Amin Shantu, El-Bayoumy Karam
Department of Biochemistry & Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA, United States of America.
Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA, United States of America.
PLoS One. 2017 Oct 26;12(10):e0186873. doi: 10.1371/journal.pone.0186873. eCollection 2017.
Genetic and epigenetic alterations observed at end stage OSCC formation could be considered as a consequence of cancer development and thus changes in normal or premalignant tissues which had been exposed to oral carcinogens such as Dibenzo[def,p]chrysene (DBP) may better serve as predictive biomarkers of disease development. Many types of DNA damage can induce epigenetic changes which can occur early and in the absence of evident morphological abnormalities. Therefore we used ERRBS to generate genome-scale, single-base resolution DNA methylomes from histologically normal oral tissues of mice treated with DBP under experimental conditions known to induce maximum DNA damage which is essential for the development of OSCC induced by DBP in mice. After genome-wide correction, 30 and 48 differentially methylated sites (DMS) were identified between vehicle control and DBP treated mice using 25% and 10% differences in methylation, respectively. RT-PCR was further performed to examine the expressions of nine selected genes. Among them, Fgf3, a gene frequently amplified in head and neck cancer, showed most prominent and significant gene expression change (2.4× increases), despite the hypomethylation of Fgf3 was identified at >10kb upstream of transcription start site. No difference was observed in protein expression between normal oral tissues treated with DBP or vehicle as examined by immunohistochemistry. Collectively, our results indicate that Fgf3 hypomethylation and gene overexpression, but not protein expression, occurred in the early stage of oral carcinogenesis induced by DBP. Thus, Fgf3 hypomethylation may serve as a potential biomarker for early detection of OSCC.
在晚期口腔鳞状细胞癌（OSCC）形成过程中观察到的基因和表观遗传改变可被视为癌症发展的结果，因此，暴露于二苯并[def,p]芘（DBP）等口腔致癌物的正常或癌前组织中的变化，可能更适合作为疾病发展的预测生物标志物。多种类型的DNA损伤可诱导表观遗传变化，这些变化可能在早期发生，且不存在明显的形态学异常。因此，我们使用酶促限制性内切酶法（ERRBS），在已知能诱导最大DNA损伤的实验条件下，从经DBP处理的小鼠组织学正常口腔组织中生成全基因组范围、单碱基分辨率的DNA甲基化图谱，这种最大DNA损伤对于DBP诱导小鼠发生OSCC至关重要。在进行全基因组校正后，分别使用甲基化差异25%和10%，在载体对照小鼠和DBP处理小鼠之间鉴定出30个和48个差异甲基化位点（DMS）。进一步进行逆转录聚合酶链反应（RT-PCR）以检测9个选定基因的表达。其中，成纤维细胞生长因子3（Fgf3）是一种在头颈癌中经常扩增的基因，尽管在转录起始位点上游>10kb处发现Fgf3存在低甲基化，但它显示出最显著和明显的基因表达变化（增加2.4倍）。通过免疫组织化学检测，在经DBP或载体处理的正常口腔组织之间未观察到蛋白质表达差异。总体而言，我们的结果表明，在DBP诱导的口腔癌发生早期出现了Fgf3低甲基化和基因过表达，但蛋白质表达未出现变化。因此，Fgf3低甲基化可能作为早期检测OSCC的潜在生物标志物。
