METTL3 in chromium-induced angiogenesis and carcinogenesis

众所周知，接触六价铬[Cr(VI)]会诱发肺癌。尽管出现了新的兴趣 在铬(VI)致癌机制中，铬(VI)在诱导RNA修饰致癌中的作用 是未知的。我们的初步研究发现，甲基转移酶样3(METTL3)的水平较高 在铬(VI)暴露小鼠的肺组织中，铬(VI)暴露的不吸烟工人的样本，以及铬(VI)- 转化的(铬-T)细胞。为了研究其可能的机制，我们发现METTL3是由上调诱导的 Nrf2和Sox4是两个重要的转录因子。我们发现METTL3在调节肌酸-睾酮中起着重要作用 细胞增殖、管状形成和肿瘤血管生成。METTL3诱导缺氧诱导因子1 )的表达，提示METTL3/PHD2/HIF-1信号转导可能在 六价铬致癌。C-X-C基序趋化因子5(CXCL5)和IL-8是METTL3的下游效应分子。 我们对暴露于铬(VI)的血液单个核细胞(PBMC)的全基因组表达阵列分析 非吸烟者和对照组中，METTL3、CXCL5和IL-8含量最高 用RT-qPCR法和ELISA法验证了铬(VI)暴露组小鼠肝细胞表面分子表达上调。我们 假设长期接触铬(VI)可诱导METTLE3过度表达以调节细胞 METTL3/PHD2/HIF-1轴在肺上皮细胞转化、肿瘤生长和血管生成中的作用 细胞，Sox4和NRF-2是两个关键的上游诱导因子。为了验证这一假设，我们将执行 目的1)确定METTL3上调在铬(VI)诱导的细胞中的作用和机制 肿瘤转化、肿瘤生长和血管生成的关系及METTL3基因上游调控因子S的研究 CR-T细胞。目的2)研究铬(VI)诱导的细胞中METTL3的关键下游靶点和分子 转化和肿瘤生长。目的3)检测肌萎缩侧索硬化症患者T细胞中METTL3的上调是否诱导 利用人源化嵌合肿瘤模型，通过CXCR1/2受体和旁分泌作用实现肿瘤血管生成； 测定NRF2、SOX4、METTL3、PHD2、HIF-1、CXCL5和/或IL-8的表达水平 铬(VI)染毒小鼠外周血单个核细胞(PBMCs)和肺组织 职业性铬(VI)接触者外周血单个核细胞我们将使用一种分子组合 方法、动物模型以及来自人类受试者和小鼠的血液和组织样本来确定这一角色 Nrf2和Sox4在中介细胞中诱导新的METTL3/PDH2/HIF-1轴的机制 转化、肿瘤生长和血管生成，并确定与体内铬(VI)的可能相关性 工人和小鼠的暴露剂量通过这些分子的水平进行。我们还将调查 METTL3/PDH2/HIF-1轴下游效应分子及其受体在Cr-T细胞诱导血管生成中的作用。 这些研究将有助于我们了解铬(VI)诱导肿瘤生长和 血管生成，并确定新的生物标志物，以早期检测铬(VI)暴露和癌症预防。

Exposure of hexavalent chromium [Cr(VI)] is known to induce lung cancer. Although there is emerging interest in mechanisms of Cr(VI)-induced carcinogenesis, role of Cr(VI) in inducing RNA modification in carcinogenesis is unknown. Our preliminary studies found that higher levels of methyltransferase like 3 (METTL3) were found in lung tissues from Cr(VI)-exposed mice, samples from Cr(VI)-exposed nonsmoking workers, and Cr(VI)- transformed (Cr-T) cells. To study underlying mechanism, we found that METTL3 was induced by upregulation of Nrf2 and SOX4, two important transcription factors. We found that METTL3 was important in regulating Cr-T cell proliferation, tube formation and tumor angiogenesis. METTL3 induced hypoxia-inducible factor 1 (HIF- 1) expression through suppressing PHD2, suggesting METTL3/PHD2/HIF-1 signaling would be important in Cr(VI) carcinogenesis. C-X-C motif chemokine 5 (CXCL5) and IL-8 were downstream effectors of METTL3. Our whole genome expression array analysis of blood mononuclear cells (PBMCs) from Cr(VI)-exposed nonsmoking workers and control subjects showed that METTL3, CXCL5 and IL-8 were among the most upregulated molecules in Cr(VI) exposure group, which was validated using RT-qPCR and ELISA assays. We hypothesize that long-term Cr(VI) exposure induces METTLE3 overexpression to regulate cell transformation, tumor growth and angiogenesis through METTL3/PHD2/HIF-1 axis in lung epithelial cells, and SOX4 and NRF-2 are two key upstream inducers. In order to test this hypothesis, we will perform three specific aims: Aim 1) To determine role and mechanism of METTL3 upregulation in Cr(VI)-induced cell transformation, tumor growth and angiogenesis, and to identify upstream regulator(s) of METTL3 elevation in Cr-T cells. Aim 2) To investigate key downstream targets and molecules of METTL3 in Cr(VI)-induced cell transformation and tumor growth. Aim 3) To determine whether METTL3 upregulation in Cr-T cells induces tumor angiogenesis through CXCR1/2 receptors and paracrine effect using humanized chimeric tumor model; to determine expression levels of Nrf2, SOX4, METTL3, PHD2, HIF-1, CXCL5, and/or IL-8 in peripheral blood mononuclear cells (PBMCs) and lung tissues from the Cr(VI)-exposed mice and in PBMCs from workers with occupational exposure to Cr(VI). We will use a combination of molecular approaches, animal models, and blood and tissue samples from human subjects and mice to define the role and mechanisms of new METTL3/PDH2/HIF-1 axis induced by Nrf2 and SOX4 in mediating cell transformation, tumor growth and angiogenesis, and determine the possible correlations with Cr(VI) internal exposure doses in workers and in mice via levels of these molecules. We will also investigate the effects of downstream effectors of METTL3/PDH2/HIF-1 axis, and their receptors in Cr-T cell-inducing angiogenesis. These studies will help us understand underlying mechanisms of Cr(VI) in inducing tumor growth and angiogenesis, and identify new biomarkers for early detection of Cr(VI) exposure and cancer prevention.