MEG3 deletion drives lung tumorigenesis due to environmental nickel exposure

摘要 几十年的流行病学研究和体外和体内实验室研究一直都是 据报道，环境中的镍暴露与肺癌之间存在关联。早在1990年， 国际癌症研究机构(IARC)将镍化合物归类为第一类致癌物质： 被确认为对人类致癌的物质。尽管包括氧化在内的多种机制 应激、表观遗传效应和触发差异基因调控的信号通路的激活 一直被认为与镍的致癌作用有关，没有证据表明单一的镍- 据我们所知，诱导因子可以驱动人类支气管细胞的转化。在我们的 初步研究，我们使用Illumina进行了长非编码RNA(LncRNA)的深度测序 HiSeqTM2000/2500高通量系统评估镍是否影响5929的丰度 已知的InncRNA。镍处理改变了正常原始人中24个LncRNAs的水平 其中16株经荧光定量聚合酶链式反应(Real-Time-PCR)鉴定。使用 对这16个LncRNAs的过表达和/或敲除分析，我们进一步发现 母体表达基因3(MEG3)的丰度足以使HBECs恶性转化。 我们证明MEG3抑制可以独立地转化HBECs，这为我们的 核心假设MEG3下调驱动HBECs的转化和致瘤性 镍暴露。在这里，我们打算阐明支撑我们新发现的分子机制。 MEG3下调是镍诱导HBECs恶性转化的关键驱动因素 具体目标如下：1、检验p62和C-Myc定义一个重要的MEG3调节基因的假设 在MEG3缺乏时促进HBECs转化的轴；2，检验假设 P62/C-Myc级联和PHLPP/HIF-1α轴之间的串扰导致MEG3缺乏- HBECs的相关恶变；3、探讨MEG3缺失的生物学意义 及其激活的下游分子在肺肿瘤发生中的作用。我们新的初步发现表明 MEG3在体外和体内都受到镍暴露的下调，仅MEG3的敲除就可以 转化HBEC，以及MEG3下游假定的信号转导之间的串扰 介导MEG3缺乏所致的HBECs恶变。这项提议的优点是 细胞培养模型和新的条件性MEG3基因敲除小鼠模型的互补应用 检查说明镍介导的肺癌发生的分子事件的整合。 澄清这些问题将提供关于MEG3作为预后生物标志物和/或作为 一个治疗靶点。最终，这两种用途都可以改善肺癌患者的临床结果。

Abstract Decades of both epidemiological research and in vitro and in vivo laboratory studies have consistently reported an association between environmental nickel exposure and lung cancer. As early as 1990, the International Agency for Research on Cancer (IARC) classified nickel compounds as Group 1 carcinogens: substances confirmed as carcinogenic to humans. Although the multiple mechanisms, including oxidative stress, epigenetic effects, and activation of signaling pathways that trigger differential gene regulation has been thought to be associated with nickel carcinogenic effect, there is no evidence that a single nickel- inducible factor can drive transformation of human bronchial cells, to the best of our knowledge. In our Preliminary Studies, we performed long noncoding RNA (lncRNA) deep sequencing using the Illumina HiSeqTM2000/2500 high throughout system to evaluate whether nickel affects the abundance of 5,929 known lncRNAs. Nickel treatment altered the levels of 24 of these lncRNAs in normal primary human bronchial epithelial cells (HBECs), while 16 of them were verified in Real-time PCR assay. With overexpression and/or knockdown analyses of the 16 lncRNAs, we further discovered that a reduced abundance of maternally-expressed gene 3 (MEG3) was sufficient for malignant transformation of HBECs. Our demonstration that MEG3 inhibition can independently transform HBECs provides the basis of our central hypothesis that MEG3 downregulation drives transformation and tumorigenecity of HBECs after nickel exposure. Here we propose to elucidate the molecular mechanisms that underlie our novel findings that MEG3 downregulation is a crucial driver for nickel-induced malignant transformation of HBECs with the following Specific Aims: 1, To test the hypothesis that p62 and C-Myc define an important MEG3-regulated axis that promotes transformation of HBECs upon MEG3 deficiency; 2, To test the hypothesis that the crosstalk between a p62/C-Myc cascade and the PHLPP/HIF-1α axis causes the MEG3-deficiency- correlated malignant transformation of HBECs; 3, To explore the biological significance of MEG3 deletion and its activated downstream molecules in lung tumoriginecity. Our novel Preliminary Findings suggest that MEG3 is downregulated by nickel exposure both in vitro and in vivo, that knockdown of MEG3 alone can transform HBECs, and that crosstalk between the putative signaling transducers downstream of MEG3 mediate the malignant transformation of HBECs caused by MEG3 deficiency. This proposal's strengths are the complementary use of cell culture models and novel conditional MEG3 knockout mouse models to examine integration of the molecular events that account for nickel-mediated lung carcinogenesis. Clarifying these issues will provide valuable insights regarding MEG3 as a prognostic biomarker and/or as a therapeutic target. Ultimately, both uses could improve clinical outcomes in lung cancer patients.