MicroRNA-190 and Oxidative Stress in Arsenic carcinogenesis

描述（由申请人提供）：我们以前的研究已经清楚地表明，三价砷（As 3+）可以诱导细胞转化。本申请的目的是确定miR-190在As 3+诱导的支气管上皮细胞恶性转化和肺肿瘤发生中的作用。主要重点将放在ROS和Erk介导的miR-190表达和miR-190依赖性Akt激活和肿瘤发生上。我们的初步数据显示（a）As 3+以剂量和时间依赖的方式诱导人支气管上皮细胞系BEAS-2 B和人小气道上皮细胞系SAEC中miR-190的产生;（B）miR-190通过下调PHLPP的合成介导As 3+诱导的Akt活化，PHLPP是Akt信号传导的内源性抑制剂和肿瘤抑制剂;和（c）miR-190的过表达引起细胞转化。基于这些初步研究，我们假设As 3+诱导的miR-190负责持续的Akt激活，随后是细胞转化，从而导致肿瘤发生。为了验证这一假设，提出了三个具体目标：具体目标1将研究As 3+如何在BEAS-2B和SAEC细胞系中诱导miR-190。我们将重点研究丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）家族中的关键成员Erk对miR-190前体转录和成熟的调控作用，特异性目的2将确定As 3+诱导的活性氧（reactive oxygen species，ROS）在As 3+激活Erk和miR-190产生中的作用。我们将鉴定As 3+诱导的每一种活性氧，并确定As 3+诱导的激活Erk和诱导miR-190的ROS的来源;具体目标3将使用过表达和原位肿瘤发生策略来研究miR-190，ROS，Erk和Akt在As 3+诱导的致癌作用中的作用，通过过表达miR-190，抗氧化酶，以及shRNA介导的人支气管上皮细胞中Erk或Akt的沉默。我们将使用稳定的转染子，并确定过表达miR-190，抗氧化酶和Erk或Akt沉默对基础或As 3+诱导的细胞转化和致癌作用的影响，通过软琼脂中的锚定非依赖性生长（集落形成）和裸鼠肺中细胞的接种试验。本项目的完成将建立As 3+诱导miR-190产生的机制及其在As 3+致癌中的作用。

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.