MiR-30a and miR-379 modulate retinoic acid pathway by targeting DNA methyltransferase 3B in oral cancer.
口腔癌中 MiR-30a 和 miR-379 通过靶向 DNA 甲基转移酶 3B 调节维甲酸途径。
- 作者列表："Shiah SG","Hsiao JR","Chang HJ","Hsu YM","Wu GH","Peng HY","Chou ST","Kuo CC","Chang JY
BACKGROUND: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. METHODS: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. RESULTS: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. CONCLUSIONS: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.
背景: 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) 癌变的发病机制和临床结局有关。然而，OSCC 中 RA 信号相关基因异常沉默的确切机制尚未得到很好的研究。 方法: 采用 40 对匹配的 OSCC 正常-肿瘤标本的全基因组基因表达和甲基化谱的联合分析,我们发现一组维甲酸信号相关基因在 OSCC 患者样本中经常高甲基化和下调，包括乙醇脱氢酶、含铁 1 (ADHFE1) 和醛脱氢酶 1 家族。成员 A2 (ALDH1A2),它们是合成 RA 的重要限速酶。采用实时定量 PCR (qRT-PCR) 和免疫组织化学方法检测口腔鳞癌患者 ADHFE1 和 ALDH1A2 的表达。使用一系列生物信息学工具预测 miR-30a 和 miR-379 与 DNA 甲基转移酶 3B (DNMT3B) 的结合位点，并使用双荧光素酶测定和 Western blot 分析进行验证。MiR-30a 、 miR-379 和 DNMT3B 的功能通过生长和菌落形成分析使用增益和功能丧失方法进行访问。通过槟榔碱和 4-(甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮 (NNK) 处理，进行染色质免疫沉淀 (ChIP)，探讨其分子机制。 结果: 我们证明了失调的 miR-30a 和 miR-379 可能是通过靶向 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 的再表达，并导致口腔癌细胞的生长抑制。此外，暴露于吸烟和咀嚼槟榔可能导致 miRNAs 和 DNMT-3B 的失调。 结论: 我们的研究结果表明，吸烟和咀嚼槟榔可以抑制 miR-30a 和 miR-379，从而上调 DNMT3B 的表达，进而导致 ADHFE1 和 ALDH1A 基因的高甲基化,促进致癌活性。这些发现强调了类视黄醇与表观遗传修饰因子联合用于预防或治疗口腔癌的潜力。
METHODS:INTRODUCTION:Human papillomavirus (HPV) is the most common sexually transmitted infection and is associated with several types of cancer. The number of cases of HPV-associated head and neck squamous cell carcinomas (HNSCCs), especially oropharyngeal carcinomas, has increased significantly in recent years despite decreased tobacco smoking rates. Currently, no data concerning the risk factors and prevalence of HPV in HNSCC patients in all regions of Brazil are available, making it difficult to promote advances in this field of public health. Therefore, our goal is to determine the impact of infection by HPV, including HPVs with different genotypes, on head and neck cancer and the risk factors associated with the development of head and neck cancer in all regions of Brazil. METHODS AND ANALYSIS:This is a case-control study that will include 622 patients and 622 controls from all regions of Brazil. A questionnaire will be applied to gather information on sociodemographic, behavioural and health factors. Oral, cervical or penile/scrotal, and anal specimens and serum samples will be collected from all participants. Formalin-fixed paraffin-embedded tissue from tumour biopsies will be analysed only in the case group. Molecular and serological analyses will be performed to evaluate the presence and role of HPV in the development of head and neck cancer. ETHICS AND DISSEMINATION:This project was approved by the research ethical committee of the proposing institution (Hospital Moinhos de Vento, number 2.852.060). Ethical approval from the collaborators is currently under evaluation and is not yet complete. The results of this study will be presented at meetings with the Brazilian Ministry of Health through technical reports and to the scientific community at national and international events, with subsequent publication of scientific articles.
METHODS:BACKGROUND:Factors related to head and neck cancer and the treatment of the disease can affect quality of life. The aim of this study was to determine factors associated with the severity of impact on oral health-related quality of life (OHRQoL) in survivors of head and neck cancer using a multivariate analysis. METHODS:This cross-sectional study evaluated 90 volunteers who had completed radiotherapy at least 3 months earlier. OHRQoL was assessed using oral health impact profile (OHIP-14) and the data were analyzed using robust variance poisson regression models. RESULTS:The mean total OHIP-14 score was 23.98 ± 12.55. Patients with hyposalivation had 56% higher (worse) mean OHIP-14 total scores (CI:1.11-2.18) and patients with advanced stage tumors had 31% higher mean OHIP-14 total scores (CI:1.03-1.66) in multivariate analyses. CONCLUSION:OHRQoL of survivors of head and neck cancer experienced a negative impact following radiotherapy. The impact was associated with hyposalivation and advanced stage tumors.
METHODS:BACKGROUND:To immunohistochemically evaluate the association between the presence of cancer-associated fibroblasts (CAFs) and the tumour expression of podoplanin (PDPN) in head and neck squamous cell carcinoma (HNSCC) and their association with clinicopathological variables. MATERIAL AND METHODS:A tissue microarray (TMA) with biopsy sections from patients diagnosed with HNSCC was stained with antibodies against the CAFs marker, α-smooth muscle actin (α-SMA), and PDPN. We subsequently evaluated their expression to determine the association between them and with clinicopathological variables including age, primary tumour site, TNM stage, and tumour differentiation grade. RESULTS:Positive reaction to α-SMA was observed in the tumour stroma, revealing spindle-shaped cells compatible with CAFs, which showed a high expression in 62% of cases and a significant association with laryngeal carcinomas, advanced clinical stages, and lower tumour differentiation (P ≤ 0.05). PDPN staining on tumour cells showed low expression in 72% of cases, and it was not associated with any clinicopathological variable or with the presence of CAFs. CONCLUSIONS:The presence of CAFs in the tumour stroma is related to an aggressive phenotype and could increase as the disease progresses, although based on our findings, it would have no relationship, at least directly, with the expression of PDPN.