ases up to 5-fold three days after endoderm priming of undifferentiated hESC line H1. Our transcriptome analysis revealed that over-expression of miR-27 in human embryonal carcinoma cells leads to downregulation of pluripotency-associated genes, such as GDF3, LIN28, TRIM71, DNMT3A, DNMT3B and USP46 and an activated expression of developmental genes such as SMAD6, BMP2, FST and HAND1. We observed an increased expression of LEFTY2, a gene that has been PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19717433 previously reported to be abundantly expressed in hESC. However, LEFTY2 is a key factor in various developmental processes and a previous knockdown study from our group reported an up-regulated expression of LEFTY2 after siRNA mediated knockdown of OCT4 or NANOG in the embryonal carcinoma cell line NCCIT. Moreover, it has been recently reported that the NODAL inhibitor, LEFTY2, is down-regulated by miR-302, a microRNA that is highly enriched in hESC, thus revealing that modulating LEFTY2 at the translational level might be important to promote the undifferentiated stage. More strikingly, we observed an up-regulation of genes that control developmental pathways such as p53-, WNT- and TGF-signalling after miR-27 over-expression in NCCIT cells. Finally, we have shown that overexpression of miR-27 in hEC leads to a dramatic reduction in expression of OCT4 mRNA and protein but, as shown with the eGFP-sensor approach, OCT4 is not a direct target gene of miR-27. The fact that loss of OCT4 Saracatinib biological activity induces activation of miR-27 expression in hES and that miR-27 over-expression results in reduced OCT4 expression in hEC, might imply 
that OCT4 and miR-27 form an indirect negative feedback loop but OCT4 rather than miR-27, is required for the maintenance of self-renewal in pluripotent stem cells as depicted in Conclusion In summary, we have demonstrated Over expression of miR-27 in hEC leads to a downregulation of OCT4 and LIN28 on the transcriptional and translational level. Loss of OCT4 expression and function in hES results in the induction of miR-27 expression. miR-27 directly targets a number of pluripotency-associated genes such as TGFR1, ACVR2, SMAD2, LIN28B, POLR3G, NR5A2 and NANOG. Therefore, we postulate that miR-27, a negatively regulated OCT4 target, is an inhibitor of self-renewal in hEC. However, both OCT4 and miR-27, operate in a larger differentiation mechanism that involves a negative feedback loop. Validation of this hypothesis is beyond the scope of this manuscript. Material and Methods Cell culture HEK293 and NCCIT cells were cultured in high-glucose DMEM supplemented with 10% FCS and 2 mM glutamine. For differentiation assays, NCCIT cells were cultured in the presence of 10 mM retinoic acid or 2 mM SB431542 and the medium was changed daily over a period of one week. hESC line H1 were grown on BD Matrigel-coated plates in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19717786 mouse embryonic fibroblast -conditioned medium containing 8 ng/ml bFGF. miR-27 Negatively Regulates Pluripotency-Associated Genes in hEC Cells Validation of miR-27 target genes In order to search for miR-27 target genes, we used miRNA target gen prediction web tools such as TargetScan, miRanda, DIANA – microT-CDS and miRWalk. PCR fragments flanking the predicted miR-27 binding sites were cloned into the 39-UTR of the modified EGFP-C1 vector. Primer sequences that have been used to generate EGFP-sensor constructs were designed using Primer3 program and analysed in BLAST for specificity. Validation of miR-27 target genes was performed as previously described. Therefore, 56104
