Inhibition of miR-221/222 in differentiating GICs increases Nestin expression and decreases GFAP and TUJ1 levels.

<p>miR-221 and miR-222 inhibition in GN1C cells growing in differentiation medium was carried out with specific anti-miRs and was confirmed by q-RT-PCR 14 days after transfection, compared to cells transfected with anti-miR negative control (anti-C-) (A, B). The expression of miR-221 and miR-222 was normalized with respect to RNU6B as 2^-ΔCt. The expression of the progenitor marker Nestin (NES) as well as of astrocytic (GFAP) and neuronal (TUJ1) differentiation markers was also measured by q-RT-PCR and normalized with respect to GAPDH and to the cells transfected in parallel with the anti-miR negative control as 2^-ΔΔCt (C, D). Transfections were carried out in triplicate. Dotted lines indicate the expression level of GN1C cells transfected with anti-miR negative control. The corresponding protein expression levels of Nestin, GFAP and TUJ1 were visualized by immunofluorescence (E) using antibodies conjugated to FITC (green) or Texas Red (red) counterstained with DAPI (blue). Images were acquired at 20X magnification with a 739CCD camera coupled to an Axio Imager Z1 microscope (Carl Zeiss Inc.) using the Isis Imaging System software. Quantification of mean intensity for Nestin, GFAP and TUJ1 fluorescence is displayed (F). * indicates p value <0.05 using unpaired t-test or Mann-Whitney U test for statistical analysis and the Holm-Bonferroni correction for multiple comparisons. </p

Over-expression of miR-29a/29b targets <i>MCL1</i> and promotes apoptosis in GICs.

<p>miR-29a/b over-expression in GN1C cells transfected with pre-miR-29a (pre-29a) or pre-miR-29b (pre-29b) compared to pre-miR negative control (pre-C-) was confirmed by q-RT-PCR 4 days after transfection (A, B). Cell viability assays using MTS (C) and apoptosis assessment by Cell Death Detection kit (D) were carried out 4 days after transfection. MCL1 protein levels were also measured by Western blot at the same time point, using β-Actin as loading control (E). Quantification of Western blots was performed with ImageJ (F) and is displayed as the MCL1/β-actin ratio relative to the negative control (100%). Regulation of the 3´-UTR of <i>MCL1</i> by miR-29a and miR-29b was analyzed by luciferase assays (G). All experiments were carried out at least in triplicate. * indicates p value <0.05 in unpaired t test or Mann-Whitney U test, using the Holm-Bonferroni correction for multiple comparisons.</p

The NS cultures are enriched in GICs when compared to the glioblastoma cell line U87MG.

<p><i>In </i><i>vitro</i> self-renewal limiting dilution assays, performed in 12 wells per dilution in triplicate experiments (A), and clonogenic assays, performed in quadruplicate 96-well plates (B) for three NS cell lines: G63, G52 and GN1C; as well as for the U87MG cell line, used as a negative control, are depicted. * indicates statistical p value <0.05 using unpaired t-test with the Holm-Bonferroni correction for multiple comparisons. Tumors obtained from <i>in </i><i>vivo</i> xenografts (1x10⁶ cells injection) in the brain striatum of BALB/c-Rag2^-/--IL2γc^-/- mice (n=6 per cell line) were detected using microPET and are shown circled by a dotted line (C). Tumor images corresponding to G63, G52 and G97C xenografts, as well as a negative control brain, are displayed, along with the corresponding quantification of maximum value of standardized caption (SUV_max). A coronal section (200 mm thick) of a tumor originated by G97C was observed by means of a stereoscopic microscope (D) for tumor localization (black arrowhead) and a semi-thin section of the tumor was stained with hematoxylin-eosin (E).</p

miR-21 over-expression in GICs induces GFAP expression, decreases Nestin levels and targets <i>SPRY1</i>.

<p>miR-21 over-expression was analyzed by q-RT-PCR 7 days after transfection and growth in NS medium (A). 2^-ΔCt was calculated as miR-21 expression relative to RNU6B expression for GN1C cells transfected with pre-miR negative control (pre-C-) or miR-21 precursor (pre-21). mRNA expression levels of Nestin (NES) as well as astrocytic (GFAP) and neuronal (TUJ1) differentiation markers were measured by q-RT-PCR (B). 2^-ΔΔCt was calculated relative to GAPDH expression and GN1C cells transfected with pre-miR negative control (dotted lines). Transfections were carried out in triplicate. Protein expression levels of Nestin, GFAP and TUJ1 were visualized by immunofluorescence (C), 7 days after transfection of GN1C cells with miR-21 precursor (pre-21) or pre-miR negative control (pre-C-). Images were acquired at 20X magnification with a 739CCD camera coupled to an Axio Imager Z1 microscope (Carl Zeiss Inc.) using the Isis Imaging System software. Quantification of mean intensity for Nestin, GFAP and TUJ1 fluorescence is displayed (D). SPRY1 and β-Actin (loading control) protein levels were measured by Western blot in the GN1C and G63 cell lines at the NS state (0h) and after 96 hours (96h) of induction of their differentiation (E), as well as 96 hours after transfection with pre-miR-21 (21) or a pre-miR negative control (C-) (F). A time-course study of SPRY protein expression by Western blot was also performed at 48, 72 and 96 hours after transfection with pre-miR-21 (21) or a pre-miR negative control (C-) (G). Quantification of at least three independent replicates was performed using ImageJ software and is shown as SPRY1/β-Actin expression relative to pre-miR negative control transfected cells (100%). Specific binding of miR-21 to the 3´-UTR binding sites of <i>SPRY1</i> was assessed in the GN1C cell line using luciferase assays and site-directed mutagenesis (H) “Mut 1” corresponds to mutation of the site in position chr4:124324121-124324128 and “Mut 2” to chr4:124323893-124323900. Transfections were carried out in triplicate. * indicates p value <0.05 in unpaired t test statistical analysis using the Holm-Bonferroni correction for multiple comparisons. </p

Microarray profiling of miRNA expression during NS differentiation.

<p>A heat map of a hierarchical clustering analysis of the microRNAs with differential expression between the NS cell lines at their basal state (NS) and after 4 (4d) and 14 (14d) days of induction of differentiation is displayed (A). Expression data are represented as log2Ratio and were mean centered for each miRNA. Validation of the differential expression of miR-29a (B), miR-29b (C), miR-221 (D), miR-222 (E), miR-21 (F), miR-93 (G) and miR-106a (H) was carried out by q-RT-PCR using specific TaqMan microRNA assays, normalizing their expression values with respect to RNU6B levels and to the NS state by calculating 2^-ΔΔCt. * indicates statistical p value <0.05 using unpaired t-test with the Holm-Bonferroni correction for multiple comparisons; dotted lines indicate the basal expression level at the NS state.</p

Homeobox NKX2-3 promotes marginal-zone lymphomagenesis by activating B-cell receptor signalling and shaping lymphocyte dynamics

NKX2 homeobox family proteins have a role in cancer development. Here we show that NKX2-3 is overexpressed in tumour cells from a subset of patients with marginal-zone lymphomas, but not with other B-cell malignancies. While Nkx2-3-deficient mice exhibit the absence of marginal-zone B cells, transgenic mice with expression of NKX2-3 in B cells show marginal-zone expansion that leads to the development of tumours, faithfully recapitulating the principal clinical and biological features of human marginal-zone lymphomas. NKX2-3 induces B-cell receptor signalling by phosphorylating Lyn/Syk kinases, which in turn activate multiple integrins (LFA-1, VLA-4), adhesion molecules (ICAM-1, MadCAM-1) and the chemokine receptor CXCR4. These molecules enhance migration, polarization and homing of B cells to splenic and extranodal tissues, eventually driving malignant transformation through triggering NF-κB and PI3K-AKT pathways. This study implicates oncogenic NKX2-3 in lymphomagenesis, and provides a valid experimental mouse model for studying the biology and therapy of human marginal-zone B-cell lymphomas