Epigenetic silencing of retinoic acid (RA) signaling-related genes have been linked with the pathogenesis and clinical outcome in oral squamous cell carcinoma (OSCC) carcinogenesis. However, the precise mechanisms underlying the abnormal silencing of RA signaling-related genes in OSCC have not been well investigated. Using combined analysis of genome-wide gene expression and methylation profile from 40 matched normal-tumor pairs of OSCC specimens, we found a set of retinoid signaling related genes are frequently hypermethylated and downregulated in OSCC patient samples, including alcohol dehydrogenase, iron containing 1 (ADHFE1) and aldehyde dehydrogenase 1 family, member A2 (ALDH1A2), which are the important rate-limiting enzymes in synthesis of RA. The expression of ADHFE1 and ALDH1A2 in OSCC patients was determine by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. The binding sites of miR-30a and miR-379 with DNA methyltransferase 3B (DNMT3B) were predicted using a series of bioinformatic tools, and validated using dual luciferase assay and Western blot analyses. The functions of miR-30a, miR-379, and DNMT3B were accessed by growth and colony formation analyses using gain- and loss-of-function approaches. Chromatin immunoprecipitation (ChIP) was performed to explore the molecular mechanisms by arecoline and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) treatment. We demonstrated that deregulated miR-30a and miR-379 could represent a mechanism for the silencing of ADHFE1 and ALDH1A2 in OSCC through targeting DNMT3B. Ectopic expression of miR-30a and miR-379 could induce re-expression of methylation-silenced ADHFE1 and ALDH1A2, and lead to growth inhibition in oral cancer cells. Furthermore, the dysregulation of the miRNAs and DNMT-3B may result from exposure to tobacco smoking and betel quid chewing. Our results demonstrate that tobacco smoking and betel quid chewing could repress miR-30a and miR-379, which upregulate the DNMT3B expression, in turn, lead to the hypermethylation of ADHFE1 and ALDH1A genes, consequently, promote the oncogenic activity. These findings highlight the potential use of retinoids in combination with epigenetic modifiers for the prevention or treatment of oral cancer.

中文翻译：

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MiR-30a和miR-379通过靶向口腔癌中的DNA甲基转移酶3B来调节视黄酸途径。

维甲酸（RA）信号相关基因的表观遗传沉默与口腔鳞状细胞癌（OSCC）癌变的发病机理和临床结果有关。但是，尚未深入研究OSCC中RA信号相关基因异常沉默的确切机制。使用来自40对匹配的正常肿瘤对OSCC标本的全基因组基因表达和甲基化谱图的组合分析，我们发现OSCC患者样品中的一组类视色素信号相关基因经常被超甲基化并下调，包括酒精脱氢酶，含铁1（ ADHFE1）和醛脱氢酶1家族成员A2（ALDH1A2），它们是RA合成中重要的限速酶。通过定量实时PCR（qRT-PCR）和免疫组织化学测定OSCC患者中ADHFE1和ALDH1A2的表达。使用一系列生物信息学工具预测了miR-30a和miR-379与DNA甲基转移酶3B（DNMT3B）的结合位点，并使用双重荧光素酶测定法和Western blot分析进行了验证。miR-30a，miR-379和DNMT3B的功能通过功能获得和丧失功能的生长和菌落形成分析获得。进行了染色质免疫沉淀（ChIP），以探索槟榔碱和4-（甲基亚硝胺基）-1-（3-吡啶基）-1-丁酮（NNK）处理的分子机制。我们证明，解除调节的miR-30a和miR-379可能代表通过靶向DNMT3B沉默OSCC中ADHFE1和ALDH1A2的机制。miR-30a和miR-379的异位表达可诱导甲基化沉默的ADHFE1和ALDH1A2的重新表达，并导致口腔癌细胞的生长受到抑制。此外，miRNA和DNMT-3B的失调可能是由于吸烟和槟榔咀嚼所致。我们的结果表明，吸烟和槟榔咀嚼可以抑制miR-30a和miR-379，从而上调DNMT3B的表达，进而导致ADHFE1和ALDH1A基因的高度甲基化，从而促进致癌活性。这些发现凸显了类视色素与表观遗传修饰剂联合用于预防或治疗口腔癌的潜在用途。暴露于吸烟和槟榔咀嚼可能会导致miRNA和DNMT-3B的失调。我们的结果表明，吸烟和槟榔咀嚼可以抑制miR-30a和miR-379，从而上调DNMT3B的表达，进而导致ADHFE1和ALDH1A基因的高度甲基化，从而促进致癌活性。这些发现凸显了类视色素与表观遗传修饰剂联合用于预防或治疗口腔癌的潜在用途。暴露于吸烟和槟榔咀嚼可能会导致miRNA和DNMT-3B的失调。我们的结果表明，吸烟和槟榔咀嚼可以抑制miR-30a和miR-379，从而上调DNMT3B的表达，进而导致ADHFE1和ALDH1A基因的高度甲基化，从而促进致癌活性。这些发现凸显了类视色素与表观遗传修饰剂联合用于预防或治疗口腔癌的潜在用途。