Kusumoto Takanobu, Hamada Tomofumi, Yamada Norishige, Nagata Satoshi, Kanmura Yuji, Houjou Izumi, Kamikawa Yoshiaki, Yonezawa Suguru, Sugihara Kazumasa
Department of Maxillofacial Diagnostic and Surgical Science, Field of Oral and Maxillofacial Rehabilitation, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.
J Oral Maxillofac Surg. 2012 Jun;70(6):1486-94. doi: 10.1016/j.joms.2011.04.021. Epub 2011 Aug 6.
To prove that chromatin immunoprecipitation assay can be performed with oral rinse samples and to develop a protocol for comprehensive analysis of functional interactions among DNA methylation, histone modification, and gene expression using such samples.
Eleven cancer cell lines and oral rinse samples from 10 patients with oral squamous cell carcinoma and 3 healthy subjects were examined. The expression of CDKN2A, a tumor suppressor gene, was determined by reverse transcription/polymerase chain reaction and immunohistochemistry. Promoter DNA methylation was assessed by methylation-specific polymerase chain reaction. Chromatin modifications were analyzed by a chromatin immunoprecipitation assay using antibodies for dimethylation and acetylation of lysine 9 of histone H3.
Epigenetic control of CDK2NA was observed in vitro in 11 cancer cell lines. Using the present protocol, comprehensive epigenetic analysis could be successfully performed with oral rinse samples. All patients were comfortable using the prescribed amount (16 mL) of normal saline to rinse their mouths. Nine patients (90%) and 1 healthy subject (33%) showed dimethylation of lysine 9 of histone H3. Moreover, 8 patients (80%) showed hypoacetylation of lysine 9 of histone H3, which was not observed in healthy subjects.
The present study showed for the first time that chromatin modifications can be analyzed using oral rinse samples by chromatin immunoprecipitation analysis. To evaluate the contribution of histone modifications for carcinogenesis of oral squamous cell carcinoma, studies including a larger number of subjects should be conducted in the future.
证明可使用口腔冲洗样本进行染色质免疫沉淀分析，并制定一种使用此类样本对DNA甲基化、组蛋白修饰和基因表达之间的功能相互作用进行综合分析的方案。
检测了11种癌细胞系以及来自10例口腔鳞状细胞癌患者和3名健康受试者的口腔冲洗样本。通过逆转录/聚合酶链反应和免疫组织化学测定肿瘤抑制基因CDKN2A的表达。通过甲基化特异性聚合酶链反应评估启动子DNA甲基化。使用针对组蛋白H3赖氨酸9二甲基化和乙酰化的抗体，通过染色质免疫沉淀分析来分析染色质修饰。
在11种癌细胞系中体外观察到CDK2NA的表观遗传调控。使用本方案，可成功地对口腔冲洗样本进行全面的表观遗传分析。所有患者使用规定量（16 mL）的生理盐水漱口都很舒适。9例患者（90%）和1名健康受试者（33%）显示组蛋白H3赖氨酸9二甲基化。此外，8例患者（80%）显示组蛋白H3赖氨酸9低乙酰化，而在健康受试者中未观察到。
本研究首次表明，可通过染色质免疫沉淀分析使用口腔冲洗样本分析染色质修饰。为了评估组蛋白修饰对口腔鳞状细胞癌致癌作用的贡献，未来应开展包括更多受试者的研究。
