Nur77 promotes cell viability and proliferation.

<p>(A) Daoy cells were transduced with pSIN-Nur77 (Nur77) or pSIN vector (EV), and cell viability was measured via the CellTiter-Glo assay every day for 4 days. Viability for each day was normalized to that of Day 0 (0 hours), and statistical significance was calculated for each day. (B) Cells were stained with crystal violet every day for 4 days to measure proliferation over time. The absorbance was measured and normalized to that of Day 0 (0 hours). The statistical significance was calculated for each day. (C) Cell proliferation was monitored by using an IncuCyte live-cell imager for real-time imaging. The resulting cell confluence was recorded every 12 hours for 4 days. (D) Nur77 mRNA level was measured after transduction with Nur77. All experiments were performed by using Daoy cells transduced with EV or Nur77 lentivirus. All data shown are representative of 3 independent experiments; *<i>p</i>≤ 0.0001.</p

Overview of Nur77 regulation by miR-124.

<p>Nur77 can be directly targeted by miR-124, as revealed by our studies reported here (indicated by the red line), or indirectly affected by miR-124 via Sp1. Nur77 may act through several downstream target genes to promote cell proliferation and survival.</p

Regulation of Nuclear Receptor Nur77 by miR-124

<div><p>The nuclear receptor Nur77 is commonly upregulated in adult cancers and has oncogenic functions. Nur77 is an immediate-early response gene that acts as a transcription factor to promote proliferation and protect cells from apoptosis. Conversely, Nur77 can translocate to the mitochondria and induce apoptosis upon treatment with various cytotoxic agents. Because Nur77 is upregulated in cancer and may have a role in cancer progression, it is of interest to understand the mechanism controlling its expression. MicroRNAs (miRNAs) are responsible for inhibiting translation of their target genes by binding to the 3ʹUTR and either degrading the mRNA or preventing it from being translated into protein, thereby making these non-coding endogenous RNAs vital regulators of every cellular process. Several miRNAs have been predicted to target Nur77; however, strong evidence showing the regulation of Nur77 by any miRNA is lacking. In this study, we used a luciferase reporter assay containing the 3ʹUTR of Nur77 to screen 296 miRNAs and found that miR-124, which is the most abundant miRNA in the brain and has a role in promoting neuronal differentiation, caused the greatest reduction in luciferase activity. Interestingly, we discovered an inverse relationship in Daoy medulloblastoma cells and undifferentiated granule neuron precursors in which Nur77 is upregulated and miR-124 is downregulated. Exogenous expression to further elevate Nur77 levels in Daoy cells increased proliferation and viability, but knocking down Nur77 via siRNA resulted in the opposite phenotype. Importantly, exogenous expression of miR-124 reduced Nur77 expression, cell viability, proliferation, and tumor spheroid size in 3D culture. In all, we have discovered miR-124 to be downregulated in instances of medulloblastoma in which Nur77 is upregulated, resulting in a proliferative state that abets cancer progression. This study provides evidence for increasing miR-124 expression as a potential therapy for cancers with elevated levels of Nur77.</p></div

miR-124 decreases cell proliferation in 2D and 3D cultures.

<p>(A) Expression of miR-124 was significantly (<i>p</i> < 0.0001) increased after antibiotic selection of Daoy cells transduced with pEZX-MR03-miR-124. As a result, Nur77 mRNA levels were significantly decreased (<i>p</i> < 0.0001). Data shown are the average of 6 independent experiments. (B) The CellTiter-Glo assay was used to analyze the cell viability of Daoy cells stably expressing exogenous miR-124 or vector control (MR03). Viability for each day was normalized to that of Day 0 (0 hours), and statistical significance was calculated for each day; *<i>p</i> < 0.0001. (C) Stable cells were imaged by using the IncuCyte live-cell imager to determine cell proliferation over the course of 3.5 days, and statistical significance was determined for each day; *<i>p</i> < 0.0001. (D) Parental Daoy cells (Daoy) and Daoy cells stably expressing exogenous miR-124 (miR-124) or its control vector (MR03) were seeded at 3 densities (288, 800, and 2500 cells/well) and grown using 3D culture techniques. After 23 days (left panel), the cells’ spheroid areas (*<i>p</i> < 0.01) were measured by using the IN Cell Analyzer (middle panel). Viability (*<i>p</i> < 0.05) was determined by performing CellTiter-Glo 3D Cell Viability Assays and is shown as raw luminescence units (RLU) (right panel). The spheroid area data shown are for cells seeded at an initial density of 800 cells per well. Data from B are representative of 5 independent experiments; data from C are representative of 4 independent experiments, and data from D are representative of 2 independent experiments.</p

Nur77 knockdown decreases cell viability and proliferation.

<p>(A) Daoy cells were transfected with 20 nM siNur77 or non-targeting control (NT), and cell viability was measured via the CellTiter-Glo assay every day for 3 days. Viability for each day was normalized to that of Day 0 (0 hours), and statistical significance was calculated for each day; *<i>p</i> < 0.0001. (B) Cells were stained with crystal violet every day for 3 days to measure proliferation over time. The absorbance was measured and normalized to that of Day 0 (0 hours). The statistical significance was calculated for each day; *<i>p</i> < 0.01. (C) Proliferation was monitored via the IncuCyte live-cell imager. Cell confluence was averaged, with 4 replicates of each condition; *<i>p</i> < 0.0001. (D) Nur77 mRNA was significantly (<i>p</i> < 0.0001) decreased after transfecting Daoy cells with siNur77. (E) Images shown for each NT and siNur77 panel over 5 days are the same image view within the same well and are representative of 3 independent experiments with 4 wells for each condition. These images correspond to the data in C. Data shown in A are representative of 5 independent experiments; data in B are representative of 4 independent experiments, and data in C and E are representative of 2 independent experiments. Data shown in D is the average of 4 independent experiments.siNur77, SMARTpool siNur77 (Catalog # M-003426-04) from GE Healthcare.</p

miR-124 decreases Nur77 levels.

<p>(A) Endogenous expression levels of miR-124 and Nur77 (mRNA) were measured in Daoy cells and human cortical neurons (HCN-2). (B) Nur77 (mRNA) and miR-124 expression are inversely related in Daoy cells exogenously expressing various levels of miR-124. The levels of Nur77 and miR-124 changed in an inversely correlated manner. (C) Daoy cells were co-transfected with the Nur77-3ʹUTR reporter plasmid (Nur77-3ʹUTR-Luc) and either an inhibitor of miR-124 (10–160 nM of oligonucleotide used as indicated) (mirVana inhibitor from Life Technologies) or an oligo control (Cntrl) (Life Technologies); resulting luciferase levels were measured. The data shown are representative of 3 independent experiments. (D) Either miR-124 or the control vector (MR03) was exogenously expressed in Daoy cells, and the resulting levels of miR-124 and Nur77 (mRNA) were measured along with the expression of Nur77 target genes. (E) Daoy cells were co-transfected with miR-124 (124) or vector control (MR03) and Flag-Nur77 plasmid with (3ʹUTR) or without (Nur77) the 3ʹUTR to confirm that miR-124 targets the 3ʹUTR. Flag and actin protein levels were detected by Western blot and quantified by using ImageJ. Levels of Flag protein were first normalized to those of actin; then MR03-3UTR was set to 1, and all other samples were compared to this sample. The Western blot shown is representative of 3 independent experiments, and the bar graph shows the average protein fold-change from 3 experiments. * indicates <i>p</i> < 0.05.</p