MicroRNA miR-378 Regulates Nephronectin Expression Modulating Osteoblast Differentiation by Targeting GalNT-7 Shireen Kahai1,5., Shao-Chen Lee1,5., Daniel Y. Lee1,5., Jennifer Yang 2 , Minhui Li1,3,5, Chia-Hui Wang1,5, Zide Jiang 3 , Yaou Zhang 4 , Chun Peng 2 , Burton B. Yang1,5* 1 Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Canada, 2 Department of Biology, York University, Toronto, Canada, 3 Department of Plant Pathology, South China Agricultural University, Guangzhou, China, 4 Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen, China, 5 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Abstract MicroRNAs (miRNAs) are small fragments of single-stranded RNA containing 18-24 nucleotides, and are generated from endogenous transcripts. MicroRNAs function in post-transcriptional gene silencing by targeting the 39-untranslated region (UTR) of mRNAs, resulting in translational repression. We have developed a system to study the role of miRNAs in cell differentiation. We have found that one of the miRNAs tested in our system (miR-378, also called miR-378*) plays a role in modulating nephronectin-mediated differentiation in the osteoblastic cell line, MC3T3-E1. Nephronectin is an extracellular matrix protein, and we have demonstrated that its over-expression enhanced osteoblast differentiation and bone nodule formation. Furthermore, we found that the nephronectin 39-untranslated region (39UTR) contains a binding site for miR-378. Stable transfection of MC3T3-E1 cells with miR-378 inhibited cell differentiation. MC3T3-E1 cells stably transfected with nephronectin exhibited higher rates of differentiation and nodule formation as compared with cells transfected with nephronectin containing the 39UTR in the early stages of development, suggesting that endogenous miR-378 is present and active. However, in the later stages of MC3T3-E1 development, the differentiation rates were opposite, with higher rates of differentiation and nodule formation in the cells over-expressing the 39UTR of nephronectin. This appeared to be the consequence of competition between nephronectin and UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalac- tosaminyltransferase 7 (GalNAc-T7 or GalNT7) for miR-378 binding, resulting in increased GalNT7 activity, which in turn lead to increased nephronectin glycosylation and product secretion, thereby resulting in a higher rate of osteoblast differentiation. Citation: Kahai S, Lee S-C, Lee DY, Yang J, Li M, et al. (2009) MicroRNA miR-378 Regulates Nephronectin Expression Modulating Osteoblast Differentiation by Targeting GalNT-7. PLoS ONE 4(10): e7535. doi:10.1371/journal.pone.0007535 Editor: Stefan W o¨lfl, Universita¨t Heidelberg, Germany Received February 12, 2009; Accepted September 28, 2009; Published October 21, 2009 Copyright: 2009 Kahai 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 work was supported by grants from Heart and Stroke Foundation of Ontario (NA 6282) and National Sciences and Engineering Research Council of Canada (227937-01) to BBY, who is also the recipient of a Career Investigator Award (CI 5958) from the Heart and Stroke Foundation of Ontario, a China-Canada Joint Health Research Grant (from Canadian Institutes of Health Research and the National Natural Science Foundation of China to BBY and YZ), and a grant from Canadian Institutes of Health Research (MOP89931) to CP who is the recipient of a Mid-Career Award from OWHC/CIHR. DYL is the recipient of a Canada Graduate Scholarship. JY was supported by an NSERC Summer Student Award. 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: byang@sri.utoronto.ca . These authors contributed equally to this work. Introduction Over the past few years, microRNAs (miRNAs) have emerged as a prominent class of gene regulatory factors [1]. MiRNAs are single-stranded RNAs of 18–24 nucleotides in length, and are generated from endogenous transcripts producing hairpin struc- tures by an RNase III-type enzyme [2–5]. miRNA functions as a regulator in gene silencing by partially complementing with the 39- untranslated region (39UTR) of target mRNAs, leading to translational repression [6–8]. By silencing various target mRNAs, miRNAs have key roles in various regulatory pathways. This involves cell proliferation [9,10], division [11,12], apoptosis [13,14], cell differentiation [15–20], tissue development [21–27], tumor formation [28–43], protein expression [44–46], immuno- response [47], and viral infection [48–51]. Although miRNAs have emerged as key regulators of gene expression, our understanding of the specific roles of miRNAs has been limited due to the difficulty in tracking the functions of a particular miRNA. Furthermore, since chemically synthetic miRNAs are easily degraded, it is impossible to obtain stable cell lines expressing miRNAs for long-term functional analysis in vitro and in vivo. Although expression of a large DNA fragment has made stable expression possible [52], in many cases miRNAs are expressed as a cluster, making it difficult to distinguish the function of a particular miRNA from others. To allow long-term studies of miRNA functions in vitro and in vivo, we have developed an expression vector expressing two copies of pre-miRNAs, a green fluorescent protein (GFP) tracking unit, and an antibiotic selection marker [53,54]. This allows stable expression of double amounts of the miRNA of interest in cells for functional studies. PLoS ONE | www.plosone.org 1 October 2009 | Volume 4 | Issue 10 | e7535