An integrated analysis of the SOX2 microRNA response program in human pluripotent and nullipotent stem cell lines

Vencken et al. BMC Genomics 2014, 15:711 http://www.biomedcentral.com/1471-2164/15/711 RESEARCH ARTICLE Open Access An integrated analysis of the SOX2 microRNA response program in human pluripotent and nullipotent stem cell lines Sebastian F Vencken1,2*, Praveen Sethupathy3, Gordon Blackshields1,2, Cathy Spillane1,2, Salah Elbaruni1,2, Orla Sheils1,2, Michael F Gallagher1,2*† and John J O’Leary1,2† Abstract Background: SOX2 is a core component of the transcriptional network responsible for maintaining embryonal carcinoma cells (ECCs) in a pluripotent, undifferentiated state of self-renewal. As such, SOX2 is an oncogenic transcription factor and crucial cancer stem cell (CSC) biomarker in embryonal carcinoma and, as more recently found, in the stem-like cancer cell component of many other malignancies. SOX2 is furthermore a crucial factor in the maintenance of adult stem cell phenotypes and has additional roles in cell fate determination. The SOX2-linked microRNA (miRNA) transcriptome and regulome has not yet been fully defined in human pluripotent cells or CSCs. To improve our understanding of the SOX2-linked miRNA regulatory network as a contribution to the phenotype of these cell types, we used high-throughput differential miRNA and gene expression analysis combined with existing genome-wide SOX2 chromatin immunoprecipitation (ChIP) data to map the SOX2 miRNA transcriptome in two human embryonal carcinoma cell (hECC) lines. Results: Whole-microRNAome and genome analysis of SOX2-silenced hECCs revealed many miRNAs regulated by SOX2, including several with highly characterised functions in both cancer and embryonic stem cell (ESC) biology. We subsequently performed genome-wide differential expression analysis and applied a Monte Carlo simulation algorithm and target prediction to identify a SOX2-linked miRNA regulome, which was strongly enriched with epithelial-to-mesenchymal transition (EMT) markers. Additionally, several deregulated miRNAs important to EMT processes had SOX2 binding sites in their promoter regions. Conclusion: In ESC-like CSCs, SOX2 regulates a large miRNA network that regulates and interlinks the expression of crucial genes involved in EMT. Keywords: SOX2, microRNA, Embryonic stem cell, Embryonal carcinoma, Pluripotency, EMT Background SOX2 is a member of the SRY-related HMG-box (SOX) transcription factor family with a set of well-established and diverse roles in stem cell potency and maintenance, embryonic development and cancer [1-10]. It regulates extensive and often divergent transcriptional networks across different cell types [1,2,11,12]. SOX2 is best known as a core pluripotency factor, maintaining the * Correspondence: ; † Equal contributors 1 Department of Histopathology, Trinity College Dublin, Sir Patrick Dun Research Laboratory, St. James’s Hospital, Dublin, Ireland 2 The Coombe Women and Infants University Hospital, Dublin, Ireland Full list of author information is available at the end of the article undifferentiated phenotype of pluripotent stem cells, and is closely co-regulated alongside core pluripotency factors OCT4 and NANOG in undifferentiated embryonic stem cells (ESCs), embryonal carcinoma cells (ECCs) and induced pluripotent stem cells (iPSCs) [8,13,14]. Loss of SOX2 expression in these cell lines triggers their differentiation. More recently, SOX2 has been identified as a crucial player in the maintenance and differentiation of adult stem cells such as in neural stem cells [15]. As an oncogene, SOX2 has been implicated in many different malignancies of the central nervous, gastrointestinal, circulatory, respiratory, endocrine and skeletal systems, and also those of the skin, liver, gonads and breast © 2014 Vencken et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Vencken et al. BMC Genomics 2014, 15:711 http://www.biomedcentral.com/1471-2164/15/711 [1-7,10]. However, despite its oncogenic potential in numerous tumour types, the suppression of SOX2 has been reported as a hallmark of gastric carcinoma [16-18]. In malignancy growing evidence reveals SOX2 to be a central regulator of a tumourigenic, stem cell-like subpopulation of tumour cells, frequently referred to as cancer stem cells (CSCs), which are found to be responsible for the proliferative and invasive capacities of most tumour types [7]. Many of the genes regulated by SOX2 in normal stem cells are aberrantly regulated by this transcription factor in cancerous cells with a similar, albeit malignant phenotype. MicroRNAs (miRNA) are a functional family of short (21–23 nt), non-protein coding RNA transcripts that primarily, but not exclusively, confer regulation of gene expression by targeting mRNAs for degradation or transient translational repression by post-transcriptionally binding these in a directed manner [19]. They are involved in the regulation of almost all cell processes and maintain cell homeostasis in both healthy and disease conditions. In cancer and CSCs, many miRNAs have been identified as tumour suppressive or oncogenic miRNAs (oncomiRs) [20]. Many of these miRNAs also have important regulatory functions in pluripotent cells, such as ESCs and ECCs, and in embryonic development [20,21]. Although the SOX2 transcriptome of protein coding genes has been previously mapped in various cell types and tissues, including ECCs, with techniques such as chromatin immunoprecipitation (ChIP) and gene array profiling, no extensive SOX2-transcriptome analysis has been performed for miRNAs in human pluripotent cells [1,2,11,12,22-25]. Some insight into the SOX2-linked miRNAome in murine pluripotent cells has previously been provided by Marson et al. who performed an extensive ChIP-sequencing (ChIP-seq) analysis of SOX2-bound miRNA promoters in mouse ESCs [26]. Additionally, in a study of the SOX2 regulatory network in human ESCs (hESCs), Boyer et al. produced a limited set of exclusively intragenic miRNAs that were potentially regulated by the SOX2-binding sites within the promoter regions of their respective host genes [11]. However, both Marson et al. and Boyer et al. provide no SOX2 knock-down and miRNA expression analysis to functionally link this transcription factor to specific miRNAs. Fang et al. profiled genes and miRNAs regulated by SOX2 in glioblastoma multiforme (GBM) cells [1]. Notably absent from this study were an in-depth analysis of miRNAs directly regulated by SOX2 and a large scale combinatorial study of the gene target regulome of deregulated miRNAs in these cells. To map the functions of this miRNA netwo (...truncated)