National Institute of Cancer Research, National Health Research Institutes, No. 35 Keyan Road, Zhunan Town, Miaoli County, 35053, Taiwan.
Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan.
J Exp Clin Cancer Res. 2019 Jun 28;38(1):281. doi: 10.1186/s13046-019-1283-z.
Discoidin domain receptor-1 (DDR1) tyrosine kinase is highly expressed in a variety of human cancers and involved in various steps of tumorigenesis. However, the precise mechanisms underlying the abnormal expression of DDR1 in oral squamous cell carcinoma (OSCC) has not been well investigated.
The expression of DDR1 on OSCC patients was determine by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Specific targeting by miRNAs was determined by software prediction, luciferase reporter assay, and correlation with target protein expression. The functions of miR-486-3p and DDR1 were accessed by MTT and Annexin V analyses using gain- and loss-of-function approaches. Chromatin immunoprecipitation (ChIP) and methylation specific PCR (MSP) were performed to explore the molecular mechanisms by arecoline treatment.
Here, we reported that DDR1 was significantly upregulated in OSCC tissues and its levels were inversely correlated with miR-486-3p expression. The experimental results in vitro confirmed that miR-486-3p decreased DDR1 expression by targeting the 3'-UTR of DDR1 mRNA. Overexpression of miR-486-3p led to growth inhibition and apoptosis induction with a similar function by knockdown of DDR1. Aberrant methylation of ANK1 promoter was a highly prevalent in OSCC and contributes to oral carcinogenesis by epigenetic silencing of ANK1 and miR-486-3p. We found that miR-486-3p can be transcriptionally co-regulated with its host gene ANK1 through epigenetic repression. DNA methylation inhibitor treatment re-expressed ANK1 and miR-486-3p. Importantly, arecoline, a major betel nut alkaloid, recruited DNMT3B binding to ANK1 promoter for DNA methylation and then attenuated the expression of miR-486-3p in OSCC.
This study was the first to demonstrate that betel nut alkaloid may recruit DNMT3B to regulate miR-486-3p/DDR1 axis in oral cancer andmiR-486-3p and DDR1 may serve as potential therapeutic targets of oral cancer.
Discoidin domain receptor-1（DDR1）酪氨酸激酶在多种人类癌症中高度表达，并参与肿瘤发生的各个步骤。然而，DDR1 在口腔鳞状细胞癌（OSCC）中的异常表达的确切机制尚未得到很好的研究。
通过定量实时 PCR（qRT-PCR）和免疫组织化学检测 OSCC 患者 DDR1 的表达。通过软件预测、荧光素酶报告测定和与靶蛋白表达的相关性来确定 miRNA 的特异性靶向作用。通过 gain-和 loss-of-function 方法，使用 MTT 和 Annexin V 分析来评估 miR-486-3p 和 DDR1 的功能。通过用 arecoline 处理进行染色质免疫沉淀（ChIP）和甲基化特异性 PCR（MSP）来探索分子机制。
在这里，我们报道 DDR1 在 OSCC 组织中显著上调，其水平与 miR-486-3p 的表达呈负相关。体外实验结果证实，miR-486-3p 通过靶向 DDR1 mRNA 的 3'UTR 降低 DDR1 的表达。DDR1 的过表达导致生长抑制和凋亡诱导，其功能与 DDR1 的敲低相似。ANK1 启动子的异常甲基化在 OSCC 中非常普遍，通过 ANK1 和 miR-486-3p 的表观遗传沉默促进口腔癌发生。我们发现 miR-486-3p 可以通过表观遗传抑制与它的宿主基因 ANK1 一起被转录调控。DNA 甲基化抑制剂处理可重新表达 ANK1 和 miR-486-3p。重要的是，槟榔碱是槟榔果中的主要生物碱之一，它可募集 DNMT3B 结合到 ANK1 启动子上进行 DNA 甲基化，从而减弱 OSCC 中 miR-486-3p 的表达。
本研究首次证明槟榔碱可能募集 DNMT3B 来调节口腔癌中的 miR-486-3p/DDR1 轴，miR-486-3p 和 DDR1 可能作为口腔癌的潜在治疗靶点。
