Role for microRNAs in regulating glucocorticoid response and resistance in multiple myeloma

Abstract

Glucocorticoids (GCs) are widely used in the treatment of hematological malignancies such as multiple myeloma. However, the development of resistance to GCs limits their clinical utility. Response to GCs is dependent on an active glucocorticoid receptor, GR-α, expressed at wild-type levels in the GC-sensitive cell line (MM.1S). GC-resistant derivative cell lines MM.1Re and MM.1RL display significant downregulation of GR-α transcripts. In this study, we report that a luciferase reporter containing the 3'-UTR of GR-α is significantly repressed in MM.1R cells when compared to MM.1S cells, suggesting that one or several microRNAs that are upregulated in MM.1R maybe in part responsible for the downregulation of the GR-α transcript. To examine posttranscriptional mechanisms of GR regulation, we examined miRNAs that have complimentary binding sites in the 3'-UTR of GR-α and found miR-130b, miR-181a, and miR-636 to be differentially expressed between GC-sensitive and GC-resistant MM.1 cell lines. Overexpression of miR-130b in MM.1S cells results in decreased expression of endogenous GR protein and decreased activity of the luciferase reporter. In addition, in MM.1S cells, the downstream GC response of glucocorticoid-induced leucine zipper induction is decreased by the overexpression of miR-130b, and further miR-130b inhibits GC-induced apoptosis and causes resistance to GCs.

Fig. 1

Relative expression of GR protein in MM.1 cell lines. a A representative immunoblot for GR in untreated MM.1S, MM.1Re, and MM.1RL. Quantification by densitometry is indicated below each lane and normalized to expression levels in MM.1S cells. b Luciferase expression is decreased in GC-resistant cells. Using a GR 3′UTR reporter construct, luciferase activity is measured 48 h after transfection as described in the “Materials and Methods” Section. Activity is normalized to the relative light units measured in the MM.1S cells (relative to MM.1S, *p < 0.01)

Fig. 2

Identification of GR-specific miRNA. a MicroRNAs predicted to target GR-α. Diagram of potential seed sequence matches within the GR 3′UTR using Targetscan program. b Relative expression of endogenous miR-130b is accomplished by qRT-PCR analysis in MM.1RL cells relative to the expression in MM.1S cells (*p < 0.05). c Screening predicted miRNAs using GR-α-3′-UTR luciferase reporter assay. Luciferase activity was measured 48 h after transfection, and miRNA mimic expression is compared to cells transfected with the negative control miRNA mimic

Fig. 3

MiR-130b decreases GR expression in MM.1S cells. a Immunoblot for GR-α using MM.1S cell lysates following transfection with increasing concentrations of miR-130b mimic. Equivalent protein is loaded in each lane as demonstrated by GAPDH signal. b Luciferase assay using GR 3′UTR construct cotransfected with increasing concentrations of miRNA-130b mimic normalized to signal from cells transfected with 24 nM negative control miRNA (*p < 0.05)

Fig. 4

MiR-130b expression impacts GC responses in MM.1S cells. a Impact of miR-130b on GC-induced cytotoxicity is measured by MTS assay using MM.1S cells transfected with either 10 nM microRNA-130b mimic (solid line) or 10 nM control mimic (negative control, dashed line) and cotreated with a range of concentrations of dexamethasone. Data are normalized to the absorbance measured in control cells, and p values for all concentration points 3 nM and above are significant at p < 0.05. Average optical density levels for untreated samples are 0.53 for control mimic and 0.86 for miR-130b mimic. b Impact of miR-130b on GC-induced gene expression is assessed by qRT-PCR analysis of GILZ gene expression in MM.1S cells in the presence or absence of 100 nM dexamethasone normalized to untreated control. c Impact of miR-130b on the GC induction of apoptosis. Immunoblot of lysates from MM.1S cells transfected for 48 h as indicated with either10 nM control mimic (0) and 1 or 10 nM miR-130b mimic. Cells were treated plus or minus 100 nM dexamethasone for an additional 24 h. Apoptosis was assessed based on PARP cleavage. d Annexin staining of MM.1S cells 72 h after transfection with either 10 nM negative control mimic, 1 nM miR-130b mimic, or 10 nM miR-130b mimic and treated +/− 100 nM Dex for 48 h (relative to MM.1S control *p < 0.05)