Network-like impact of MicroRNAs on neuronal lineage differentiation of unrestricted somatic stem cells from human cord blood.

Unrestricted somatic stem cells (USSCs) represent an intrinsically multipotent CD45-negative fetal population from human cord blood. They show differentiation into neuronal cells of a dopaminergic phenotype, which express neuronal markers such as synaptophysin, neuronal-specific nuclear protein, and neurofilament and release the neurotransmitter dopamine accompanied by expression of dopaminergic key factors tyrosine hydroxylase and Nurr1 (NR4A2). MicroRNA expression analysis highlighted their importance in neural development but their specific functions remain poorly understood. Here, downregulation of a set of 18 microRNAs during neuronal lineage differentiation of unrestricted somatic stem cells, including members of the miR-17-92 family and additional microRNAs such as miR-130a, -138, -218, and -335 as well as their target genes, is described. In silico target gene predictions for this microRNA group uncovered a large set of proteins involved in neuronal differentiation and having a strong impact on differentiation-related pathways such as axon guidance and TGFß, WNT, and MAPK signaling. Experimental target validations confirmed approximately 35% of predictions tested and revealed a group of proteins with specific impact on neuronal differentiation and function including neurobeachin, neurogenic differentiation 1, cysteine-rich motor neuron protein 1, neuropentraxin 1, and others. These proteins are combined targets for several subgroups from the set of 18 downregulated microRNAs. This finding was further supported by the observed upregulation of a significant amount of predicted and validated target genes based on Illumina Beadstudio microarray data. Confirming the functional relationship of a limited panel of microRNAs and predicted target proteins reveals a clear network-like impact of the group of 18 downregulated microRNAs on proteins involved in neuronal development and function.

A data integration approach to mapping OCT4 gene regulatory networks operative in embryonic stem cells and embryonal carcinoma cells.

It is essential to understand the network of transcription factors controlling self-renewal of human embryonic stem cells (ESCs) and human embryonal carcinoma cells (ECs) if we are to exploit these cells in regenerative medicine regimes. Correlating gene expression levels after RNAi-based ablation of OCT4 function with its downstream targets enables a better prediction of motif-specific driven expression modules pertinent for self-renewal and differentiation of embryonic stem cells and induced pluripotent stem cells.We initially identified putative direct downstream targets of OCT4 by employing CHIP-on-chip analysis. A comparison of three peak analysis programs revealed a refined list of OCT4 targets in the human EC cell line NCCIT, this list was then compared to previously published OCT4 CHIP-on-chip datasets derived from both ES and EC cells. We have verified an enriched POU-motif, discovered by a de novo approach, thus enabling us to define six distinct modules of OCT4 binding and regulation of its target genes.A selection of these targets has been validated, like NANOG, which harbours the evolutionarily conserved OCT4-SOX2 binding motif within its proximal promoter. Other validated targets, which do not harbour the classical HMG motif are USP44 and GADD45G, a key regulator of the cell cycle. Over-expression of GADD45G in NCCIT cells resulted in an enrichment and up-regulation of genes associated with the cell cycle (CDKN1B, CDKN1C, CDK6 and MAPK4) and developmental processes (BMP4, HAND1, EOMES, ID2, GATA4, GATA5, ISL1 and MSX1). A comparison of positively regulated OCT4 targets common to EC and ES cells identified genes such as NANOG, PHC1, USP44, SOX2, PHF17 and OCT4, thus further confirming their universal role in maintaining self-renewal in both cell types. Finally we have created a user-friendly database (http://biit.cs.ut.ee/escd/), integrating all OCT4 and stem cell related datasets in both human and mouse ES and EC cells.In the current era of systems biology driven research, we envisage that our integrated embryonic stem cell database will prove beneficial to the booming field of ES, iPS and cancer research.

The molecular basis of genistein-induced mitotic arrest and exit of self-renewal in embryonal carcinoma and primary cancer cell lines.

BACKGROUND: Genistein is an isoflavonoid present in soybeans that exhibits anti-carcinogenic properties. The issue of genistein as a potential anti-cancer drug has been addressed in some papers, but comprehensive genomic analysis to elucidate the molecular mechanisms underlying the effect elicited by genistein on cancer cells have not been performed on primary cancer cells, but rather on transformed cell lines. In the present study, we treated primary glioblastoma, rhabdomyosarcoma, hepatocellular carcinoma and human embryonic carcinoma cells (NCCIT) with mu-molar concentrations of genistein and assessed mitotic index, cell morphology, global gene expression, and specific cell-cycle regulating genes. We compared the expression profiles of NCCIT cells with that of the cancer cell lines in order to identify common genistein-dependent transcriptional changes and accompanying signaling cascades. METHODS: We treated primary cancer cells and NCCIT cells with 50 muM genistein for 48 h. Thereafter, we compared the mitotic index of treated versus untreated cells and investigated the protein expression of key regulatory self renewal factors as OCT4, SOX2 and NANOG. We then used gene expression arrays (Illumina) for genome-wide expression analysis and validated the results for genes of interest by means of Real-Time PCR. Functional annotations were then performed using the DAVID and KEGG online tools. RESULTS: We found that cancer cells treated with genistein undergo cell-cycle arrest at different checkpoints. This arrest was associated with a decrease in the mRNA levels of core regulatory genes, PBK, BUB1, and CDC20 as determined by microarray-analysis and verified by Real-Time PCR. In contrast, human NCCIT cells showed over-expression of GADD45 A and G (growth arrest- and DNA-damage-inducible proteins 45A and G), as well as down-regulation of OCT4, and NANOG protein. Furthermore, genistein induced the expression of apoptotic and anti-migratory proteins p53 and p38 in all cell lines. Genistein also up-regulated steady-state levels of both CYCLIN A and B. CONCLUSION: The results of the present study, together with the results of earlier studies show that genistein targets genes involved in the progression of the M-phase of the cell cycle. In this respect it is of particular interest that this conclusion cannot be drawn from comparison of the individual genes found differentially regulated in the datasets, but by the rather global view of the pathways influenced by genistein treatment.