Forced Expression of miR-143 Represses ERK5/c-Myc and p68/p72 Signaling in Concert with miR-145 in Gut Min Tumors of Apc Mice Yuji Takaoka1., Yuko Shimizu1., Hitoki Hasegawa2,3, Yasuo Ouchi2, Shanlou Qiao 1 , Miki Nagahara2, Masatoshi Ichihara1, Jiing-Dwan Lee4, Koichi Adachi5, Michinari Hamaguchi3, Takashi Iwamoto1,2* 1 Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Aichi, Japan, 2 The Center for Education in Laboratory Animal Research, Chubu University, Kasugai, Aichi, Japan, 3 Division of Cancer Biology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan, 4 Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America, 5 Radioisotope Research Center Medical Division, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan Abstract Recently, miR-143 and miR-145 have been shown to belong to a subset of microRNAs whose expression is controlled by a complex of a tumor suppressor p53 and DEAD-box RNA helicase subunits p68/p72. While accumulating studies have acknowledged that both miRNAs function as tumor suppressors and are similarly regulated, evidence of their coordinated action against tumorigenesis has been poorly presented. Herein, we establish transgenic mice that express miR-143 under the control of the CAG regulatory unit. When crossbred with ApcMin/+ mice, the development of tumors in the small intestines is significantly attenuated. In the transgenic small intestine tumors, the endogenous miR-145 is also enhanced and the expression of c-Myc and p68/p72, both of which have been reported to be pivotal for gut tumor development, is suppressed, corresponding to the downregulation of ERK5. We demonstrate that the combination of miR-143 and miR-145 inhibits the expression of c-Myc in human colon cancer cells, whereas miR-145 retards that of p72. Moreover, we show the possibilities that miR-145 modulates p72 expression through its 39 untranslated region and that c-Myc downregulation is involved in both p68 suppression and miR-145 induction. These findings suggest that forced expression of miR-143, probably interacting with endogenous miR-145, inhibits ERK5/c-Myc and p68/p72/b-catenin signaling and hampers small intestine tumor development in ApcMin/+ mice. This unique cascade, in turn, may prevent overproduction of a subset of tumor suppressive miRNAs by repressing their own modulators, p68/p72. Citation: Takaoka Y, Shimizu Y, Hasegawa H, Ouchi Y, Qiao S, et al. (2012) Forced Expression of miR-143 Represses ERK5/c-Myc and p68/p72 Signaling in Concert with miR-145 in Gut Tumors of ApcMin Mice. PLoS ONE 7(8): e42137. doi:10.1371/journal.pone.0042137 Editor: Wayne A. Phillips, Peter MacCallum Cancer Centre, Australia Received February 28, 2012; Accepted July 2, 2012; Published August 2, 2012 Copyright: 2012 Takaoka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported in part by Grants-in-Aid for Scientific Research (C) (no 19590408) and Center of Excellence Project for Private Universities (no. S0801055) from the Ministry of Education, Culture, Sports, Science, and Technology. No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: iwamoto@isc.chubu.ac.jp . These authors contributed equally to this work. Introduction A considerable number of lines of evidence have shown that some sets of microRNA (miRNA) act as oncogenes or as tumor suppressors, and that their biogenesis is closely linked to cancer [1,2,3]. miR-143 and miR-145 have emerged as tumor suppressing miRNAs, particularly for colon cancers. Michael et al. initially reported that the expression of miR-143 and miR-145 was downregulated in many colorectal neoplasms, suggesting their potential action as tumorsuppresors [4]. Indeed, this notion was supported by the following studies, which revealed that the downregulation of miR-143 and miR-145 could be involved in B- cell malignancy [5] and that colon tumor cell proliferation was suppressed by transfection with miR-143 [6]. However as most of these studies used synthetic miR-143 and miR-145 mimics, their expression was transient and usually far beyond the level normally expressed in living organisms. Hence, their physiological relevance remains to be determined. Colorectal cancer is one of the most common cancers in not only the Western world but also Japan. Mutations in the Adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP) syndrome, and are usually an initial event in sporadic cancer development. APC encodes a large multidomain protein that binds to b-catenin and promotes its destruction to downregulate the Wnt signalling pathway [7,8]. ApcMin/+ mice, which were established by random mutagenesis with ethylnitrosourea, harbor a heterozygous truncating mutation at codon 850 of Apc and develop tens of small intestinal polyps when on the C57BL/6 background, while quite a few tumors develop in colons [9,10]. RNA polymerase II initially transcribes long primary miRNAs (pri-miRNAs) which are generally more than several kilo base pairs (bp) and possess a 59 cap and poly (A) tail. miRNAs mature through a multistep modification with two members of the PLoS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42137