METTL3 in chromium-induced angiogenesis and carcinogenesis

已知六价铬[Cr（VI）]的暴露会诱发肺癌。尽管人们对 在Cr（VI）诱导的致癌机制中，Cr（VI）在致癌过程中诱导RNA修饰的作用 不明我们的初步研究发现，甲基转移酶样3（胃L3）水平较高， 在来自Cr（VI）暴露小鼠的肺组织中，来自Cr（VI）暴露非吸烟工人的样品，以及Cr（VI）- 转化（Cr-T）细胞。为了研究潜在的机制，我们发现胃L3通过上调 Nrf 2和SOX 4是两个重要的转录因子。我们发现胃L3在调节Cr-T中起重要作用 细胞增殖、管形成和肿瘤血管生成。胃L3诱导的缺氧诱导因子1 β（HIF-1 α） 提示PHD 2/HIF-1信号通路在PHD 2/L3表达中起重要作用。 Cr（VI）致癌作用。C-X-C基序趋化因子5（CXCL 5）和IL-8是胃L3的下游效应物。 我们的全基因组表达阵列分析的血液单核细胞（PBMC）从铬（VI）暴露， 结果表明，胃L3、CXCL 5和IL-8在非吸烟工人和对照组中的表达最高， 在Cr（VI）暴露组中上调分子，这使用RT-qPCR和ELISA测定法进行验证。我们 假设长期Cr（VI）暴露诱导胃LE 3过表达以调节细胞 肺上皮细胞L3/PHD 2/HIF-1轴介导的转化、肿瘤生长和血管生成 细胞，SOX 4和NRF-2是两个关键的上游诱导剂。为了验证这一假设，我们将 三个具体目的：目的1）确定Cr（VI）诱导的细胞中胃L3上调的作用和机制 本发明的目的是为了研究肿瘤转化、肿瘤生长和血管生成，并鉴定肿瘤细胞中的胃L3升高的上游调节因子。 Cr-T细胞目的2）研究Cr（VI）诱导的细胞中胃L3的关键下游靶点和分子 转化和肿瘤生长。目的3）确定在Cr-T细胞中胃L3上调是否诱导 利用人源化嵌合肿瘤模型，通过CXCR 1/2受体和旁分泌效应实现肿瘤血管生成; 为了测定Nrf 2、SOX 4、胃L3、PHD 2、HIF-1 α、CXCL 5和/或IL-8在人结肠癌细胞中的表达水平， 外周血单核细胞（PBMC）和肺组织从铬（VI）暴露的小鼠和 职业暴露于Cr（VI）的工人的PBMC。我们将使用分子的组合 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，以确定 Nrf 2和SOX 4介导的细胞凋亡中新的胃L3/PDH 2/HIF-1轴的形成及其机制 转化，肿瘤生长和血管生成，并确定可能的相关性与Cr（VI）内部 通过这些分子的水平，工人和小鼠的暴露剂量。我们还将研究 在Cr-T细胞诱导的血管生成中，胃L3/PDH 2/HIF-1轴的下游效应物及其受体。 这些研究将有助于我们了解Cr（VI）诱导肿瘤生长的潜在机制， 血管生成，并确定新的生物标志物，用于早期检测Cr（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.