MicroRNA-190 and Oxidative Stress in Arsenic carcinogenesis

描述(申请人提供)：我们之前的研究已经清楚地证明了三价砷(As3+)可以诱导细胞转化。这项应用的目的是确定miR-190在As3+诱导的支气管上皮细胞恶性转化和肺肿瘤发生中的作用。主要的重点是ROS和Erk介导的miR-190的表达以及依赖miR-190的Akt激活和肿瘤发生。我们的初步数据表明：(A)As3+以剂量和时间依赖的方式诱导人支气管上皮细胞系BEAS-2B和人小气道上皮细胞系SAEC产生miR-190；(B)miR-190通过下调Akt信号的内源性抑制因子和肿瘤抑制因子PHLPP的合成而介导As3+诱导的Akt激活；以及(C)miR-190过表达导致细胞转化。基于这些初步研究，我们假设As3+诱导的miR-190负责持续的Akt激活，随后细胞转化，从而导致肿瘤的发生。为了验证这一假设，提出了三个特定的目标：特定目标1将研究As3+如何在BEAS-2B和SAEC细胞系中诱导miR-190。我们将重点研究在As3+处理的细胞中，丝裂原活化蛋白激酶(MAPK)家族的关键成员Erk对前体miR-190转录和成熟的调控，特异性目标2将确定As3+诱导的活性氧物种(ROS)在As3+激活Erk和As3+产生miR-190中的作用。我们将鉴定As3+诱导的每个活性氧物种，并确定As3+诱导的激活Erk和诱导miR-190的ROS的来源；特定目标3将使用过表达和原位肿瘤形成策略，通过过表达miR-190、抗氧化酶miR-190和shRNA介导的Erk或Akt沉默，研究miR-190、ROS、Erk和Akt在As3+诱导的致癌中的作用。我们将使用稳定的转染体，通过软琼脂中非锚定生长(集落形成)和裸鼠肺内接种细胞的实验，确定过表达miR-190、抗氧化酶和Erk或Akt沉默对基础或As3+诱导的细胞转化和致癌的影响。该项目的完成将建立As3+诱导miR-190产生的机制及其在As3+致癌中的作用。 公共卫生相关性：环境中暴露的砷，特别是三价砷，长期以来一直是一个主要的公共卫生问题。本研究将通过检验砷诱导的microRNA-190与细胞的恶性转化和肿瘤发生有关的假说来探讨砷致癌的机制。其长期目标是了解砷致癌的分子机制，并识别生物标志物，以开发早期检测、干预和预防策略。

DESCRIPTION (provided by applicant): Our previous research has clearly demonstrated that trivalent arsenic (As3+) can induce cell transformation. The goal of this application is to determine the role of miR-190 in As3+-induced malignant transformation of the bronchial epithelial cells and the tumorigenesis of the lung. Major emphasis will be on the ROS- and Erk- mediated expression of miR-190 and the miR-190-dependent Akt activation and tumorigenesis. Our preliminary data have shown that (a) As3+ induces miR-190 generation in the human bronchial epithelial cell line, BEAS-2B, and human small airway epithelial cell line, SAEC, in a manner of both dose- and time- dependent; (b) MiR-190 mediates As3+-induced Akt activation by down-regulating the synthesis of PHLPP, an endogenous inhibitor of Akt signaling and a tumor suppressor; and (c) overexpression of miR-190 causes cell transformation. Based on these preliminary studies, we hypothesize that As3+-induced miR-190 is responsible for the sustained Akt activation, followed by cell transformation and consequently, the tumorigenesis. To test this hypothesis, three specific aims are proposed: Specific Aim 1 will investigate how As3+ induces miR-190 in BEAS-2B and SAEC cell lines. We will focus on the regulation of both transcription and maturation of the precursor miR-190 by Erk, a key member of mitogen-activated protein kinase (MAPK) family, in the cells treated with As3+, Specific Aim 2 will determine the role of As3+-induced reactive oxygen species (ROS) on the activation of Erk and miR-190 production by As3+. We will identify each of the reactive oxygen species induced by As3+, and determine the sources of As3+-induced ROS that activate Erk and induce miR-190; Specific Aim 3 will use overexpression and orthotopic tumorigenesis strategies to study the role of miR-190, ROS, Erk, and Akt in As3+-induced carcinogenesis by overexpressing miR-190, the antioxidant enzymes, and shRNA- mediated silencing of Erk or Akt in the human bronchial epithelial cells. We will use stable transfectants and determine the effects of overexpressing miR-190, antioxidant enzymes, and Erk or Akt silencing on either basal or As3+-induced cell transformation and carcinogenesis by assays of anchorage-independent growth in soft agar (colony formation) and inoculation of the cells in the lung of nude mice. The completion of this project will establish the mechanism of As3+-induced miR-190 generation and its role in As3+ carcinogenesis. PUBLIC HEALTH RELEVANCE: Environmental exposure of arsenic, especially the trivalent form arsenic, has long been a major public health concern. The present study will investigate the mechanism of arsenic-induced carcinogenesis by testing the hypothesis that arsenic-induced microRNA-190 is responsible for the malignant transformation and tumorigenesis of the cells. The long-term goals are to understand molecular mechanism of arsenic-induced carcinogenesis and to identify biomarkers for developing early detection, intervention and prevention strategies.