ABSTRACT
The let-7 family of miRNAs has been shown to be crucial in many aspects of biology, from the regulation of developmental timing to cancer. The available methods to regulate this family of miRNAs have so far been mostly genetic and therefore not easily performed experimentally. Here, we describe a small molecule screen designed to identify regulators of let-7 targets in human cells. In particular, we focused our efforts on the identification of small molecules that could suppress let-7 targets, as these could serve to potentially intercede in tumors driven by loss of let-7 activity. After screening through roughly 36,000 compounds, we identified a class of phosphodiesterase inhibitors that suppress let-7 targets. These compounds stimulate cAMP levels and raise mature let-7 levels to suppress let-7 target genes in multiple cancer cell lines such as HMGA2 and MYC. As a result, these compounds also show growth inhibitory activity on cancer cells.
Subject(s)
MicroRNAs/metabolism , Small Molecule Libraries/analysis , Small Molecule Libraries/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclic AMP Response Element-Binding Protein/metabolism , Genes, Reporter , HMGA2 Protein/metabolism , High-Throughput Screening Assays , Humans , Phosphodiesterase Inhibitors/pharmacologyABSTRACT
Using a compendium of cell-state-specific gene expression data, we identified genes that uniquely define cell states, including those thought to represent various developmental stages. Our analysis sheds light on human cell fate through the identification of core genes that are altered over several developmental milestones, and across regional specification. Here we present cell-type specific gene expression data for 17 distinct cell states and demonstrate that these modules of genes can in fact define cell fate. Lastly, we introduce a web-based database to disseminate the results.
Subject(s)
Algorithms , Cell Differentiation , Cells, Cultured , Cellular Reprogramming , Gene Expression , Gene Regulatory Networks/genetics , Humans , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
It is clear that neural differentiation from human pluripotent stem cells generates cells that are developmentally immature. Here, we show that the let-7 plays a functional role in the developmental decision making of human neural progenitors, controlling whether these cells make neurons or glia. Through gain- and loss-of-function studies on both tissue and pluripotent derived cells, our data show that let-7 specifically regulates decision making in this context by regulation of a key chromatin-associated protein, HMGA2. Furthermore, we provide evidence that the let-7/HMGA2 circuit acts on HES5, a NOTCH effector and well-established node that regulates fate decisions in the nervous system. These data link the let-7 circuit to NOTCH signaling and suggest that this interaction serves to regulate human developmental progression.
Subject(s)
MicroRNAs/genetics , Neuroglia/metabolism , Pluripotent Stem Cells/metabolism , Receptors, Notch/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/genetics , Cell Line , Gene Expression Regulation, Developmental , HMGA2 Protein/genetics , HMGA2 Protein/metabolism , Humans , Immunohistochemistry , MicroRNAs/metabolism , Nervous System/cytology , Nervous System/growth & development , Nervous System/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurogenesis/genetics , Neuroglia/cytology , Neurons/cytology , Neurons/metabolism , Oligodendroglia/cytology , Oligodendroglia/metabolism , Pluripotent Stem Cells/cytology , RNA Interference , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Receptors, Notch/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/geneticsABSTRACT
Addition of a specific set of transcription factors reprograms somatic cell nuclei to a pluripotent state. Sequential addition of these factors, rather than the simultaneous exposure used in standard protocols, improves reprogramming efficiency. This sequential method favours a transition through a state with enhanced mesenchymal characteristics before driving an epithelial transformation on the way to the pluripotent state.