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Glucocorticoid-regulated microRNAs and mirtrons in human acute lymphoblastic leukemia

ABSTRACT: Glucocorticoids (GC) have a major impact on the biology of normal and malignant cells of the lymphoid lineage. This includes induction of apoptosis which is exploited in the therapy of acute lymphoblastic leukemia (ALL) and related lymphoid malignancies. MicroRNAs (miRNAs), and the related mirtrons, are ~22 nucleotide RNA molecules implicated in the control of essential biological functions including proliferation, differentiation and apoptosis. They derive from polymerase-II transcripts but whether GCs regulate miRNA/mirtron-encoding transcription units is not known. We investigated miRNA/mirtron expression and GC regulation in 8 ALL in vitro models and 13 ALL children undergoing systemic GC monotherapy using a combination of expression profiling techniques, real time RT-PCR and northern blotting to detect mature miRNAs/mirtrons and/or their precursors. We identified a GC-regulated mirtron (miR-1233) and a number of GC-regulated miRNAs, including the myeloid-specific miR-223 and the apoptosis and cell cycle arrest-inducing mir15~16 cluster. However, the response was heterogeneous in individual patients and cell lines, the extent of regulation was moderate, and functional analyses of the most promising candidates (miR223 and the miR15~16 cluster) revealed little, if any effect, on cell cycle progression and survival. Thus, although GCs affect miRNA expression in ALL cells, these regulations may not be major contributors to the anti-leukemic GC effects. Keywords: exon analysis

ORGANISM(S): Homo sapiens

PROVIDER: GSE10910 | GEO | 2009/01/16

SECONDARY ACCESSION(S): PRJNA107203

REPOSITORIES: GEO

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Project description:Glucocorticoids (GCs) are commonly used to treat patients suffering from lymphoid malignancies i.e. leukemia and multiple myeloma. Although GCs are known to be strong inducers of apoptosis in lymphoid cells, the molecular determinants of GC therapy resistance are poorly understood. Although GC treatment triggers important changes in gene expression, few studies have addressed the regulatory role of small regulatory microRNAs (miRNAs) in GC therapy response. Only recently, aberrant microRNA expression has been linked to the development of haematological malignancies and microRNAs have become master regulators of drug resistance. We identified GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate multiple genes involved in cell cycle control, cell organization and cell death in MM1S, which remain unaffected in MM1R cells. Correspondingly, GCs selectively trigger cell death in MM1S but not in MM1R. Out of 32 microRNAs responsive to GC in MM1S cells but not in MM1R cells, mir-150 was identified as the most persistent GC responsive microRNA. Furthermore, Ingenuity Pathways Analysis (IPA) revealed that ectopic transfection of a synthetic mir-150 mimics GC therapy response in MM1S cells, associated with selective changes in mRNA levels of typical GR transactivated and transrepressed target genes. Although mir-150 largely mirrors GC responsive changes in gene expression of the transcription factor Myb, GR chaperone FKBP5, cell cycle modulator proteins (IL23A, SKP2, CDKN1A), chemokine signaling proteins (CXCR4, CX3CR1, CCL3) and mTOR/UPR stress related proteins (DDIT4, TXNIP), we also observed mir-150 selective effects on transcription factors (NR3C2 (MR), Myb, Fos, Jun, C/EBP-beta, IRF4, NFE2L1, ATF3, ATF4,), chaperone molecules HSPA8, HSP90AB1), the sodium channel SCNN1G and UPR stress proteins (TRIB3, DDIT3). Remarkably, mir-150 overexpression was not able to overcome GC therapy resistance, since we could not detect GC like effects of mir-150 in GR (NR3C1) deficient MM1R cells. Altogether GC-inducible mir-150 adds a novel complex layer of regulation for fine tuning GC specific therapeutic responses in multiple myeloma.

Project description:Gene expression data of glucocorticoid resistant and sensitive acute lymphoblastic leukemia cell lines for the article: Expression, regulation and function of phosphofructo-kinase/fructose-biphosphatases (PFKFBs) in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia cells Glucocorticoids (GCs) cause apoptosis and cell cycle arrest in lymphoid cells and constitute a central component in the therapy of lymphoid malignancies, most notably childhood acute lymphoblastic leukemia (ALL). PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-2), a kinase controlling glucose metabolism, was identified by us previously as GC response gene in expression profiling analyses performed in children with ALL during initial systemic GC mono-therapy. Since deregulation of glucose metabolism has been implicated in apoptosis induction, this gene and its relatives PFKFB1, 3, and 4 were further analyzed. Expression analyses in additional ALL children, non-leukemic individuals and leukemic cell lines confirmed frequent PFKFB2 induction by GC in most systems sensitive to GC-induced apoptosis, particularly in T-ALL cells. The 3 other family members, in contrast, were not or weakly expressed (PFKFB1 and 4) or not induced by GC (PFKFB3). Conditional PFKFB2 over-expression in the CCRF-CEM T-ALL in vitro model revealed that its 2 splice variants (15A and 15B) did not have any detectable effect on survival or cell cycle progression. Moreover, neither PFKFB2 splice variant significantly affected sensitivity to, or kinetics of, GC-induced apoptosis. Our data suggest that, at least in the model system investigated, PFKFB2 is not an essential upstream regulator of the anti-leukemic effects of GC. Generation of the GC sensitive and resistant clones is described in Parson et al. FASEB J 2005 (Pubmed id 15637111). In brief GC sensitive clones were generated by limiting dilution subcloning from the GC sensitive T-ALL cell line CCRF-CEM-C7H2. To generate GC resistant clones the CCRF-CEM-C7H2 cell line was clutured in the presence of 10E-7 M dexametasone. Gene expression profiles of glucocorticoid (GC) resistant and sensitive T-ALL cells during GC treatment and corresponding control samples (cells treated with carrier control). GC induced regulation of PFKFB2 was determined in the various cell lines based on the expression intensities of the corresponding probe sets in GC treated and control samples.

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Glucocorticoid-regulated microRNAs and mirtrons in human acute lymphoblastic leukemia

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