MEG3 deletion drives lung tumorigenesis due to environmental nickel exposure

摘要 几十年的流行病学研究以及体外和体内实验室研究始终表明， 报告了环境镍暴露与肺癌之间的联系。早在1990年， 国际癌症研究机构（IARC）将镍化合物列为第1组致癌物： 被证实对人类致癌的物质。虽然多种机制，包括氧化 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支气管细胞的转化。在我们 初步研究，我们使用Illumina进行了长非编码RNA（lncRNA）深度测序， HiSeqTM 2000/2500高通量系统评估镍是否影响5，929丰度 已知的lncRNA镍处理改变了正常人中24种lncRNA的水平 支气管上皮细胞（HBECs），其中16个经Real-time PCR验证。与 通过对16种lncRNA的过表达和/或敲低分析，我们进一步发现， 母亲表达的基因3（MEG 3）的丰度足以使HBEC恶性转化。 我们证明MEG 3抑制可以独立地转化HBEC，这为我们的研究提供了基础。 中心假设MEG 3下调驱动HBEC的转化和致瘤性， 镍暴露。在这里，我们建议阐明我们的新发现的分子机制 MEG 3下调是镍诱导的HBEC恶性转化的关键驱动因素， 以下具体目的：1、验证p62和C-Myc定义重要的MEG 3调节的细胞凋亡的假设。 2.为了验证MEG 3缺乏时促进HBEC转化的轴的假设， p62/C-Myc级联和PHLPP/HIF-1α轴之间的串扰导致MEG 3缺陷- 探讨MEG 3基因缺失的生物学意义 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， MEG 3在体外和体内都受到镍暴露的下调，单独敲低MEG 3可以 转化HBEC和MEG 3下游推定信号转导子之间的串扰 介导由MEG 3缺陷引起的HBEC恶性转化。该提案的优势在于 补充使用细胞培养模型和新的条件性MEG 3敲除小鼠模型， 检查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MEG 3作为预后生物标志物和/或作为 治疗目标最终，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.