Regulation of cellular Cyclin D1 gene by arsenic is mediated through miR-2909 M. Sharma, S. Sharma, M. Arora, Deepak Kaul ⁎ Experimental Medicine & Biotechnology Department, Postgraduate Institute of Medical Education & Research, CHANDIGARH-160012, India abstract article info Article history: Accepted 21 March 2013 Available online 4 April 2013 Keywords: miR-2909 Genomics Splice-switching Arsenic Human PBMCs Arsenic through its ability to regulate genes that link cell cycle control with apoptosis has been widely recog- nized to play a crucial role in oncogenomics. However, the molecular event by which arsenic affects such genes is far from clear. Here we provide reasonably good evidence to support the view that arsenic exposure to human PBMCs (peripheral blood mononuclear cells) at low concentrations results in the over-expression of miR-2909 within these cells. This over-expressed miR-2909 was found to regulate CCND1 (Cyclin D1) gene expression, within these cells by inducing splice-switching of tumor suppresser CYLD (Cylindromatosis) gene as well as modulation of SP1 (Specificity Protein 1) activity through the repression of KLF4 (Kruppel-like factor4) expression at the translational level. Arsenic dependent regulation of AATF (Apoptosis Antagonizing Transcription factor) and BCL3 (B-cell Lymphoma 3) were also found to be modulated through its capacity to induce miR-2909 expression. Based upon these observations, a novel epigenomic pathway was proposed which may not only be useful in understanding the paradoxical role of arsenic in oncogenomics but also may even be useful in devising various strategies for the treatment/prevention of tumors induced by arsenic. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Arsenic is ubiquitous in nature and is one of the potent toxic met- alloid known to be responsible for posing various biological effects. Low dose chronic exposure to arsenic has been linked with various types of malignancies such as skin, bladder (Letašiová et al., 2012), liver, lung and adrenal carcinoma (Kligerman and Tennant, 2007; Tokar et al., 2012). Extensive studies have established that arsenic dose dependent genome response data may be responsible for its ob- served pleiotropic effects on various cellular processes like apoptosis, proliferation, inflammation and angiogenesis (Abernathy et al., 2003; Ren et al., 2011; Tchounwou et al., 2003; Zhao et al., 1997). Chronic exposure to arsenic at low concentration has been shown to induce CCND1 dependent cellular proliferation via NFkB/BCL3 pathway but the exact mechanism is not clearly understood. NFkB signaling is regulated by various modifications like ubiquitination and deubiquitination. In this context, it is interesting to note that CYLD (isoform 1), a deubiqutinating enzyme, regulates NFkB/BCL3 pathway and also the expression of this gene is up-regulated by NFkB at the transcriptional level (Jono et al., 2004). CCND1 expression is known to be inhibited by tumor suppressor, KLF4 and SP1, both of which compete for the same binding site on CCND1 promoter (Hu et al., 2009; Shie et al., 2000). In addition to the significant role played by these genes, certain miRNA (small non coding, 18–24 nucleotides) have also been shown to regulate arsenic induced oncogenicity (Beezhold et al., 2011). Re- cent studies from our laboratory revealed that cellular AATF genome encodes a novel microRNA (miR-2909) postulated to have myriad cel- lular functions (Kaul and Hussain, 2009). At this stage it is pertinent to note that an arsenic compound induces generation of ROS (reactive oxygen species) in tumor as well as normal human cells (Jomova et al., 2011). Under such oxidative stress induced DNA damaging con- ditions, ROS increases the transcriptional expression of NFkB gene (Gloire et al., 2006). Keeping in view the above mentioned facts, the current study was designed to explore the genomic basis of this intriguing mechanism involving miR-2909, CCND1, BCL3, CYLD and KLF4 through which ar- senic regulates oncogenomics. 2. Materials & methods 2.1. Bioinformatics analysis Promoter sequences for different genes were obtained from Mam- malian Promoter sequence database (http://rulai.cshl.edu/CSHLmpd2). Putative binding sites for various transcription factors were retrieved from JASPAR database (http://jaspar.cgb.ki.se) and TFSEARCH (http:// www.cbrc.jp/research/db/TFSEARCH.html) at a default threshold score of 85.0. The presence of putative target sites of miR-2909 on 3′UTR of Gene 522 (2013) 60–64 Abbreviations: PBMCs, Peripheral blood mononuclear cells; CCND1, Cyclin D1; CYLD, Cylindromatosis; SP1, Specificity Protein 1; KLF4, Kruppel-like factor4; AATF, Ap- optosis Antagonizing Transcription factor; BCL3, B-cell Lymphoma 3; NFkB, Nuclear factor kappa B; FCS, Fetal Calf Serum; miRNA, MicroRNA; As, Arsenic; ROS, Reactive ox- ygen species; IL6, Interleukin 6. ⁎ Corresponding author. Fax: +91 172 2744401. E-mail address: dkaul_24@hotmail.com (D. Kaul). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.03.058 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene