ABSTRACT
MicroRNA-138 (miR-138) is a pro-survival oncomiR for glioma stem cells. In malignant gliomas, dysregulated expression of microRNAs, such as miR-138, promotes Tumour initiation and progression. Here, we identify the ancillary role of the CCAAT/enhancer binding protein ß (C/EBPß) as a transcriptional activator of miR-138. We demonstrate that a short 158 bp DNA sequence encoding the precursor of miR-138-2 is essential and sufficient for transcription of miR-138. This short sequence includes the A-box and B-box elements characteristic of RNA Polymerase III (Pol III) promoters, and is also directly bound by C/EBPß via an embedded 'C/EBPß responsive element' (CRE). CRE and the Pol III B-box element overlap, suggesting that C/EBPß and transcription factor 3C (TFIIIC) interact at the miR-138-2 locus. We propose that this interaction is essential for the recruitment of the RNA Pol III initiation complex and associated transcription of the oncomiR, miR-138 in malignant gliomas.
Subject(s)
CCAAT-Enhancer-Binding Protein-beta/genetics , Glioma/genetics , MicroRNAs/genetics , RNA Polymerase III/metabolism , Transcription, Genetic , Animals , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Glioma/metabolism , Glioma/therapy , HEK293 Cells , Humans , Interleukin Receptor Common gamma Subunit/deficiency , Interleukin Receptor Common gamma Subunit/genetics , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Oncogenes/genetics , Protein Binding , RNA Interference , RNAi Therapeutics/methods , Xenograft Model Antitumor Assays/methodsABSTRACT
The discovery that white adipocytes can undergo a browning process to become metabolically active beige cells has attracted significant interest in the fight against obesity. However, the study of adipose browning has been impeded by a lack of imaging tools that allow longitudinal and noninvasive monitoring of this process in vivo. Here, we report a preclinical imaging approach to detect development of beige adipocytes during adrenergic stimulation. In this approach, we expressed near-infrared fluorescent protein, iRFP720, driven under an uncoupling protein-1 (Ucp1) promoter in mice by viral transduction, and used multispectral optoacoustic imaging technology with ultrasound tomography (MSOT-US) to assess adipose beiging during adrenergic stimulation. We observed increased photoacoustic signal at 720 nm, coupled with attenuated lipid signals in stimulated animals. As a proof of concept, we validated our approach against hybrid positron emission tomography combined with magnetic resonance (PET/MR) imaging modality, and quantified the extent of adipose browning by MRI-guided segmentation of 2-deoxy-2-18F-fluoro-d-glucose uptake signals. The browning extent detected by MSOT-US and PET/MR are well correlated with Ucp1 induction. Taken together, these systems offer great opportunities for preclinical screening aimed at identifying compounds that promote adipose browning and translation of these discoveries into clinical studies of humans.
Subject(s)
Adipose Tissue, Brown/diagnostic imaging , Multimodal Imaging , 3T3-L1 Cells , Adipose Tissue, Brown/cytology , Adipose Tissue, Brown/metabolism , Animals , Biological Transport , Cell Differentiation , Fluorodeoxyglucose F18/metabolism , Magnetic Resonance Imaging , Mice , Mice, Inbred BALB C , Photoacoustic Techniques , Positron-Emission TomographyABSTRACT
Increased visceral fat, rather than subcutaneous fat, during the onset of obesity is associated with a higher risk of developing metabolic diseases. The inherent adipogenic properties of human adipose-derived stem cells (ASCs) from visceral depots are compromised compared with those of ASCs from subcutaneous depots, but little is known about the underlying mechanisms. Using ontological analysis of global gene expression studies, we demonstrate that many genes involved in retinoic acid (RA) synthesis or regulated by RA are differentially expressed in human tissues and ASCs from subcutaneous and visceral fat. The endogenous level of RA is higher in visceral ASCs; this is associated with upregulation of the RA synthesis gene through the visceral-specific developmental factor WT1. Excessive RA-mediated activity impedes the adipogenic capability of ASCs at early but not late stages of adipogenesis, which can be reversed by antagonism of RA receptors or knockdown of WT1. Our results reveal the developmental origin of adipocytic properties and the pathophysiological contributions of visceral fat depots.
Subject(s)
Adipogenesis , Down-Regulation , Gene Expression Regulation, Developmental , Intra-Abdominal Fat/metabolism , Receptors, Retinoic Acid/agonists , Signal Transduction , Tretinoin/metabolism , Active Transport, Cell Nucleus/drug effects , Adipogenesis/drug effects , Adult Stem Cells/cytology , Adult Stem Cells/drug effects , Adult Stem Cells/metabolism , Adult Stem Cells/pathology , Bariatric Surgery , Benzoates/pharmacology , Cells, Cultured , Down-Regulation/drug effects , Gene Expression Profiling , Gene Expression Regulation, Developmental/drug effects , Gene Ontology , Humans , Intra-Abdominal Fat/cytology , Intra-Abdominal Fat/drug effects , Intra-Abdominal Fat/pathology , Middle Aged , Naphthalenes/pharmacology , Obesity, Morbid/metabolism , Obesity, Morbid/pathology , Obesity, Morbid/surgery , RNA Interference , Receptors, Retinoic Acid/antagonists & inhibitors , Receptors, Retinoic Acid/metabolism , Response Elements/drug effects , Signal Transduction/drug effects , Stilbenes/pharmacology , Subcutaneous Fat, Abdominal/cytology , Subcutaneous Fat, Abdominal/drug effects , Subcutaneous Fat, Abdominal/metabolism , Subcutaneous Fat, Abdominal/pathology , Up-Regulation/drug effects , WT1 Proteins/antagonists & inhibitors , WT1 Proteins/genetics , WT1 Proteins/metabolismABSTRACT
Adipose-derived stem/stromal cells (ASCs) from the anatomically distinct subcutaneous and visceral depots of white adipose tissue (WAT) differ in their inherent properties. However, little is known about the molecular identity and definitive markers of ASCs from these depots. In this study, ASCs from subcutaneous fat (SC-ASCs) and visceral fat (VS-ASCs) of omental region were isolated and studied. High-content image screening of over 240 cell-surface markers identified several potential depot-specific markers of ASCs. Subsequent studies revealed consistent predominant expression of CD10 in SC-ASCs and CD200 in VS-ASCs across 12 human subjects and in mice. CD10-high-expressing cells sorted from SC-ASCs differentiated better than their CD10-low-expressing counterparts, whereas CD200-low VS-ASCs differentiated better than CD200-high VS-ASCs. The expression of CD10 and CD200 is thus depot-dependent and associates with adipogenic capacities. These markers will offer a valuable tool for tracking and screening of depot-specific stem cell populations.
Subject(s)
Antigens, Surface/metabolism , Intra-Abdominal Fat/cytology , Stem Cells/cytology , Stem Cells/metabolism , Subcutaneous Fat/cytology , Adipogenesis , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, Surface/genetics , Biomarkers/metabolism , Gene Expression , High-Throughput Screening Assays , Humans , Immunophenotyping , Male , Mice , Neprilysin/genetics , Neprilysin/metabolism , PhenotypeABSTRACT
Malignant gliomas are the most aggressive forms of brain tumors, associated with high rates of morbidity and mortality. Recurrence and tumorigenesis are attributed to a subpopulation of tumor-initiating glioma stem cells (GSCs) that are intrinsically resistant to therapy. Initiation and progression of gliomas have been linked to alterations in microRNA expression. Here, we report the identification of microRNA-138 (miR-138) as a molecular signature of GSCs and demonstrate a vital role for miR-138 in promoting growth and survival of bona fide tumor-initiating cells with self-renewal potential. Sequence-specific functional inhibition of miR-138 prevents tumorsphere formation in vitro and impedes tumorigenesis in vivo. We delineate the components of the miR-138 regulatory network by loss-of-function analysis to identify specific regulators of apoptosis. Finally, the higher expression of miR-138 in GSCs compared to non-neoplastic tissue and association with tumor recurrence and survival highlights the clinical significance of miR-138 as a prognostic biomarker and a therapeutic target for treatment of malignant gliomas.