The involvement of microRNAs (miRNAs) in orchestrating self-renewal and differentiation of stem cells has been revealed in a number of recent studies. And while in human pluripotent stem cells, miRNAs have been directly linked to the core pluripotency network, including the cell cycle regulation and the maintenance of the self-renewing capacity, their role in the onset of differentiation in other contexts, such as determination of neural cell fate, remains poorly described. To bridge this gap, we used three model cell types to study miRNA expression patterns: human embryonic stem cells (hESCs), hESCs-derived self-renewing neural stem cells (NSCs), and differentiating NSCs. The comprehensive miRNA profiling presented here reveals novel sets of miRNAs differentially expressed during human neural cell fate determination in vitro. Furthermore, we report a miRNA expression profile of self-renewing human NSCs, which has been lacking to this date. Our data also indicates that miRNA clusters enriched in NSCs share the target-determining seed sequence with cell cycle regulatory miRNAs expressed in pluripotent hESCs. Lastly, our mechanistic experiments confirmed that cluster miR-17-92, one of the NSCs-enriched clusters, is directly transcriptionally regulated by transcription factor c-MYC.

• Keywords
• Cell cycle, Human pluripotent stem cells, Neural stem cells, miRNA sequencing, microRNA,
• MeSH
• Cell Differentiation genetics   MeSH
• Embryonic Stem Cells MeSH
• Humans MeSH
• MicroRNAs * genetics   metabolism   MeSH
• Neural Stem Cells * metabolism   MeSH
• Gene Expression Profiling MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• MicroRNAs * MeSH

Oct4-mediated reprogramming has recently become a novel tool for the generation of various cell types from differentiated somatic cells. Although molecular mechanisms underlying this process are unknown, it is well documented that cells over-expressing Oct4 undergo transition from differentiated state into plastic state. This transition is associated with the acquisition of stem cells properties leading to epigenetically "open" state that is permissive to cell fate switch upon external stimuli. In order to contribute to our understanding of molecular mechanisms driving this process, we characterised human fibroblasts over-expressing Oct4 and performed comprehensive small-RNAseq analysis. Our analyses revealed new interesting aspects of Oct4-mediated cell plasticity induction. Cells over-expressing Oct4 lose their cell identity demonstrated by down-regulation of fibroblast-specific genes and up-regulation of epithelial genes. Interestingly, this process is associated with microRNA expression profile that is similar to microRNA profiles typically found in pluripotent stem cells. We also provide extensive network of microRNA families and clusters allowing us to precisely determine the miRNAome associated with the acquisition of Oct4-induced transient plastic state. Our data expands current knowledge of microRNA and their implications in cell fate alterations and contributing to understanding molecular mechanisms underlying it.

• MeSH
• Cell Line MeSH
• Embryo, Mammalian * MeSH
• Fibroblasts cytology   metabolism   MeSH
• Humans MeSH
• MicroRNAs * biosynthesis   genetics   MeSH
• Octamer Transcription Factor-3 * biosynthesis   genetics   MeSH
• Gene Expression Regulation * MeSH
• Cellular Reprogramming Techniques * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• MicroRNAs * MeSH
• Octamer Transcription Factor-3 * MeSH
• POU5F1 protein, human MeSH Browser

MicroRNA (miRNA) sponges are RNA transcripts containing multiple high-affinity binding sites that associate with and sequester specific miRNAs to prevent them from interacting with their target messenger (m)RNAs. Due to the high specificity of miRNA sponges and strong inhibition of target miRNAs, these molecules have become increasingly applied in miRNA loss-of-function studies. However, improperly designed sponge constructs may sequester off-target miRNAs; thus, it has become increasingly important to develop a tool for miRNA sponge construct design and testing. In this study, we introduce microRNA sponge generator and tester (miRNAsong), a freely available web-based tool for generation and in silico testing of miRNA sponges. This tool generates miRNA sponge constructs for specific miRNAs and miRNA families/clusters and tests them for potential binding to miRNAs in selected organisms. Currently, miRNAsong allows for testing of sponge constructs in 219 species covering 35,828 miRNA sequences. Furthermore, we also provide an example, supplemented with experimental data, of how to use this tool. Using miRNAsong, we designed and tested a sponge for miR-145 inhibition, and cloned the sequence into an inducible lentiviral vector. We found that established cell lines expressing miR-145 sponge strongly inhibited miR-145, thus demonstrating the usability of miRNAsong tool for sponge generation. URL: http://www.med.muni.cz/histology/miRNAsong/.

• MeSH
• HEK293 Cells MeSH
• Internet * MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• MicroRNAs genetics   metabolism   MeSH
• Computer Simulation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Messenger MeSH
• MicroRNAs MeSH
• MIRN145 microRNA, human MeSH Browser

MicroRNA (miRNAs) are short noncoding RNA molecules involved in many cellular processes and shown to play a key role in somatic cell induced reprogramming. We performed an array based screening to identify candidates that are differentially expressed between dermal skin fibroblasts (DFs) and induced pluripotent stem cells (iPSCs). We focused our investigations on miR-145 and showed that this candidate is highly expressed in DFs relative to iPSCs and significantly downregulated during reprogramming process. Inhibition of miR-145 in DFs led to the induction of "cellular plasticity" demonstrated by: (a) alteration of cell morphology associated with downregulation of mesenchymal and upregulation of epithelial markers; (b) upregulation of pluripotency-associated genes including SOX2, KLF4, C-MYC; (c) downregulation of miRNA let-7b known to inhibit reprogramming; and (iv) increased efficiency of reprogramming to iPSCs in the presence of reprogramming factors. Together, our results indicate a direct functional link between miR-145 and molecular pathways underlying reprogramming of somatic cells to iPSCs.

• Keywords
• Induced pluripotent stem cells, KLF4, Mesenchymal-to-epithelial transition, OCT4, Reprogramming, SOX2, c-MYC, miR-145, microRNA,
• MeSH
• Fibroblasts cytology   metabolism   MeSH
• Induced Pluripotent Stem Cells cytology   MeSH
• Kruppel-Like Factor 4 MeSH
• Humans MeSH
• MicroRNAs genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Cellular Reprogramming * genetics   MeSH
• Gene Expression Regulation MeSH
• Reproducibility of Results MeSH
• Base Sequence MeSH
• Dermis cytology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• KLF4 protein, human MeSH Browser
• Kruppel-Like Factor 4 MeSH
• MicroRNAs MeSH
• MIRN145 microRNA, human MeSH Browser

We examined the microRNAs (miRNAs) expressed in chronic lymphocytic leukemia (CLL) and identified miR-150 as the most abundant, but with leukemia cell expression levels that varied among patients. CLL cells that expressed ζ-chain-associated protein of 70 kDa (ZAP-70) or that used unmutated immunoglobulin heavy chain variable (IGHV) genes, each had a median expression level of miR-150 that was significantly lower than that of ZAP-70-negative CLL cells or those that used mutated IGHV genes. In samples stratified for expression of miR-150, CLL cells with low-level miR-150 expressed relatively higher levels of forkhead box P1 (FOXP1) and GRB2-associated binding protein 1 (GAB1), genes with 3' untranslated regions having evolutionary-conserved binding sites for miR-150. High-level expression of miR-150 could repress expression of these genes, which encode proteins that enhance B-cell receptor signaling, a putative CLL-growth/survival signal. Also, high-level expression of miR-150 was a significant independent predictor of longer treatment-free survival or overall survival, whereas an inverse association was observed for high-level expression of GAB1 or FOXP1 for overall survival. This study demonstrates that expression of miR-150 can influence the relative expression of GAB1 and FOXP1 and the signaling potential of the B-cell receptor, thereby possibly accounting for the noted association of expression of miR-150 and disease outcome.

• MeSH
• Adaptor Proteins, Signal Transducing biosynthesis   genetics   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell genetics   metabolism   MeSH
• Adult MeSH
• Forkhead Transcription Factors biosynthesis   genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• RNA, Small Interfering MeSH
• MicroRNAs genetics   MeSH
• Receptors, Antigen, B-Cell genetics   metabolism   MeSH
• Gene Expression Regulation, Leukemic genetics   MeSH
• Repressor Proteins biosynthesis   genetics   MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Aged MeSH
• Signal Transduction * physiology   MeSH
• Transfection MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• Forkhead Transcription Factors MeSH
• FOXP1 protein, human MeSH Browser
• GAB1 protein, human MeSH Browser
• RNA, Small Interfering MeSH
• MicroRNAs MeSH
• MIRN150 microRNA, human MeSH Browser
• Receptors, Antigen, B-Cell MeSH
• Repressor Proteins MeSH

Death ligands and their tumor necrosis factor receptor (TNFR) family receptors are the best-characterized and most efficient inducers of apoptotic signaling in somatic cells. In this study, we analyzed whether these prototypic activators of apoptosis are also expressed and able to be activated in human pluripotent stem cells. We examined human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) and found that both cell types express primarily TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNFR1, but very low levels of Fas/CD95. We also found that although hESC and hiPSC contain all the proteins required for efficient induction and progression of extrinsic apoptotic signaling, they are resistant to TRAIL-induced apoptosis. However, both hESC and hiPSC can be sensitized to TRAIL-induced apoptosis by co-treatment with protein synthesis inhibitors such as the anti-leukemia drug homoharringtonine (HHT). HHT treatment led to suppression of cellular FLICE inhibitory protein (cFLIP) and Mcl-1 expression and, in combination with TRAIL, enhanced processing of caspase-8 and full activation of caspase-3. cFLIP likely represents an important regulatory node, as its shRNA-mediated down-regulation significantly sensitized hESC to TRAIL-induced apoptosis. Thus, we provide the first evidence that, irrespective of their origin, human pluripotent stem cells express canonical components of the extrinsic apoptotic system and on stress can activate death receptor-mediated apoptosis.

• MeSH
• fas Receptor genetics   metabolism   MeSH
• Apoptosis drug effects   MeSH
• Cell Differentiation MeSH
• Embryonic Stem Cells cytology   drug effects   metabolism   MeSH
• CASP8 and FADD-Like Apoptosis Regulating Protein antagonists & inhibitors   genetics   metabolism   MeSH
• Harringtonines pharmacology   MeSH
• Homoharringtonine MeSH
• Protein Synthesis Inhibitors pharmacology   MeSH
• Caspase 3 genetics   metabolism   MeSH
• Caspase 8 genetics   metabolism   MeSH
• Humans MeSH
• RNA, Small Interfering genetics   metabolism   MeSH
• Pluripotent Stem Cells cytology   drug effects   metabolism   MeSH
• Cell Proliferation MeSH
• Myeloid Cell Leukemia Sequence 1 Protein genetics   metabolism   MeSH
• TNF-Related Apoptosis-Inducing Ligand genetics   metabolism   pharmacology   MeSH
• Receptors, Tumor Necrosis Factor, Type I genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Signal Transduction MeSH
• Drug Synergism MeSH
• Receptors, TNF-Related Apoptosis-Inducing Ligand genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• fas Receptor MeSH
• FAS protein, human MeSH Browser
• CASP8 and FADD-Like Apoptosis Regulating Protein MeSH
• Harringtonines MeSH
• Homoharringtonine MeSH
• Protein Synthesis Inhibitors MeSH
• Caspase 3 MeSH
• Caspase 8 MeSH
• RNA, Small Interfering MeSH
• MCL1 protein, human MeSH Browser
• Myeloid Cell Leukemia Sequence 1 Protein MeSH
• TNF-Related Apoptosis-Inducing Ligand MeSH
• Receptors, Tumor Necrosis Factor, Type I MeSH
• TNFRSF10A protein, human MeSH Browser
• TNFSF10 protein, human MeSH Browser
• Receptors, TNF-Related Apoptosis-Inducing Ligand MeSH