Identification of linc-NeD125, a novel long non coding RNA that hosts miR-125b-1 and negatively controls proliferation of human neuroblastoma cells

<div><p>ABSTRACT</p><p>The human genome contains some thousands of long non coding RNAs (lncRNAs). Many of these transcripts are presently considered crucial regulators of gene expression and functionally implicated in developmental processes in Eukaryotes. Notably, despite a huge number of lncRNAs are expressed in the Central Nervous System (CNS), only a few of them have been characterized in terms of molecular structure, gene expression regulation and function. In the present study, we identify linc-NeD125 as a novel cytoplasmic, neuronal-induced long intergenic non coding RNA (lincRNA). Linc-NeD125 represents the host gene for miR-125b-1, a microRNA with an established role as negative regulator of human neuroblastoma cell proliferation. Here, we demonstrate that these two overlapping non coding RNAs are coordinately induced during <i>in vitro</i> neuronal differentiation, and that their expression is regulated by different mechanisms. While the production of miR-125b-1 relies on transcriptional regulation, linc-NeD125 is controlled at the post-transcriptional level, through modulation of its stability.</p><p>We also demonstrate that linc-NeD125 functions independently of the hosted microRNA, by reducing cell proliferation and activating the antiapoptotic factor BCL-2.</p></div

miR-135a and miR-124 regulate the expression of the endogenous MR protein.

<p>(A) N2a cells were transfected with miR-135a (p135a) and miR-124(p124) overexpressing vectors as indicated. p135a+p124 corresponds to the transfection with an equimolar mixture of the two vectors. A representative blot is shown. (B) Western blot analysis of endogenous MR expression was quantified by densitometry, normalized to actin as loading control and expressed relatively to empty vector transfected cells. Data represent the mean from three biological samples and three technical replicates ± SE. *<i>P</i> < 0.05, <i>**P</i> < 0.05 (pairwise Student’s <i>t</i>-test). (C) Cerebellar granule neurons (6+4 DIV) were transfected with LNA antisense oligonucleotides or scramble LNA as negative control. A representative blot is shown. (D) Western blot analysis of endogenous MR expression was quantified by densitometry, normalized to tubulin and expressed relatively to scramble LNA transfected cells. Data represent the mean from four biological samples and three technical replicates ± SE. *<i>P</i> < 0.05, (pairwise Student’s <i>t</i>-test).</p

Nr3c2 reporter is regulated by miR-135a.

<p>(A) High probability miRNA target sequences in the mouse Nr3c2 3’ UTR are drawn. Candidate miRNAs are predicted by microT v 3.0, TargetScan 5.2, and PicTar algoritms. Positions of mir-135a and miR-124 target sequences in the mouse annotated Nr3c2 3’ UTR and details of miRNA/mRNA base pairing are indicated. Beneath miRNA sequences, nucleotides mutated at the level of miRNA binding sites in the mutant constructs Nr3c2 m135a (mutated at both miR-135a seed binding sites) and Nr3c2 m124 (mutated at both miR-124 seed binding sites), are indicated (nts in bold). (B) Nr3c2 luciferase reporter (Nr3c2), and mutant reporter constructs were co-transfected into Hela cells together with empty vector or miRNA expression vectors (p135a and p124). Luciferase activity was measured 24 hours post-transfection. Values are expressed relatively to the internal renilla luciferase activity and presented as percentage of the activity achieved in the presence of the empty control vector. Results are shown as means ± SE (n=6). **<i>P</i><0.005 (pairwise Student’s <i>t</i>-test).</p

Acute stress induces miR-135a and miR-124 downregulation in the amygdala.

<p>(A) The differential expression of mature miRNAs in the amygdala was evaluated by microarray analysis after 2 hours of restraint. The MA plot shows relative change values, expressed as log₂ ratio (stress vs control), plotted against average log intensity ((log₂Hy5+log₂Hy3)/2). (B) Levels of mature miRNAs are quantified in the amygdala RNA pool by qRT-PCR, using U6B as internal control. The statistical test used for comparison was one-way ANOVA (<i>n</i>=9). Values are means ± SE *<i>P</i> <0.001 versus naive control mice.</p

Acute stress increases MR protein levels in amygdala.

<p>(A) Steady state MR protein levels were measured by western blot analysis immediately after 2 hours of restraint. Lysates were obtained from pooled amygdala nuclei (<i>n</i>=4), 5 pools were generated from naive (<i>n</i>=20) and restrained mice (<i>n</i>=20). MR expression was normalized to actin signals in the same blot. Quantitative values are shown as mean ± SE <i>*P</i> < 0.05 (pairwise Student’s <i>t</i>-test). (B) qRT-PCR analysis of Nr3c2 transcript levels in naive and stressed mice (<i>n</i>=12 for both groups). Data are presented as mean ± SE. Ex 6-7 and exs 8-9 refer to the amplicons studied, corresponding to exons 6 and 7 (exs 6-7), and to exons 8 and 9 (exs 8-9) of the Nr3c2 coding sequence.</p

Mir-23a and mir-125b regulate neural stem/progenitor cell proliferation by targeting Musashi1

<div><p>Musashi1 is an RNA binding protein that controls the neural cell fate, being involved in maintaining neural progenitors in their proliferative state. In particular, its downregulation is needed for triggering early neural differentiation programs. In this study, we profiled microRNA expression during the transition from neural progenitors to differentiated astrocytes and underscored 2 upregulated microRNAs, miR-23a and miR-125b, that sinergically act to restrain Musashi1 expression, thus creating a regulatory module controlling neural progenitor proliferation.</p></div

IFN-α Regulates Blimp-1 Expression via miR-23a and miR-125b in Both Monocytes-Derived DC and pDC

<div><p>Type I interferon (IFN-I) have emerged as crucial mediators of cellular signals controlling DC differentiation and function. Human DC differentiated from monocytes in the presence of IFN-α (IFN-α DC) show a partially mature phenotype and a special capability of stimulating CD4+ T cell and cross-priming CD8+ T cells. Likewise, plasmacytoid DC (pDC) are blood DC highly specialized in the production of IFN-α in response to viruses and other danger signals, whose functional features may be shaped by IFN-I. Here, we investigated the molecular mechanisms stimulated by IFN-α in driving human monocyte-derived DC differentiation and performed parallel studies on peripheral unstimulated and IFN-α-treated pDC. A specific miRNA signature was induced in IFN-α DC and selected miRNAs, among which miR-23a and miR-125b, proved to be negatively associated with up-modulation of Blimp-1 occurring during IFN-α-driven DC differentiation. Of note, monocyte-derived IFN-α DC and <i>in vitro</i> IFN-α-treated pDC shared a restricted pattern of miRNAs regulating Blimp-1 expression as well as some similar phenotypic, molecular and functional hallmarks, supporting the existence of a potential relationship between these DC populations. On the whole, these data uncover a new role of Blimp-1 in human DC differentiation driven by IFN-α and identify Blimp-1 as an IFN-α-mediated key regulator potentially accounting for shared functional features between IFN-α DC and pDC.</p> </div

IFN-α induces a specific pattern of miRNAs during DC differentiation: <i>PRDM-1</i> is a miRNA target gene.

<p><b>A</b>. Fold induction of specific miRNAs in DC differentiated from monocytes after IFN-α treatment with respect to monocytes treated with GM-CSF alone. Each miRNA was analyzed by qRT-PCR on 10 different donors, except miR-146a, whose data were available only for 5 donors (donor 6 to 10). Broken lines indicate out of range values. <b>B</b>. miRNAs found to be up-regulated or down-regulated, with respect GM-CSF-treated monocytes, in DC differentiated in the presence of IFN-α or IL-4. Color intensities and numbers indicate the relative median of fold-change values. Fold-change threshold was fixed at ± 1.2 in at least 8 out of 10 investigated donors for IFN-α DC and in at least 4 out of 5 donors for IL-4 DC and for miR-146a in IFN-α DC. Bold type represents miRNAs whose expression changed in both IFN-α DC and IL-4 DC, but in opposite direction. <b>C</b>. Expression of miRNAs whose target site is predicted in the 3’ UTR of the Blimp-1-coding <i>PRDM-1</i> gene in DC differentiated with IFN-α or IL-4. Median fold-changes are indicated in each colored box.</p

IFN-α stimulates the expression of Blimp-1.

<p><b>A</b>. Kinetics of Blimp-1 expression in IFN-α DC was analyzed by qRT-PCR with respect to freshly isolated monocytes. Normalized data represent the mean ± SD of 4 independent experiments. Wilcoxon test was performed: *<i>p</i>= 0.0034; **<i>p</i>= 0.0005. <b>B</b>. Blimp-1 protein expression analyzed by western blot from day 2 to day 4 in IFN-α DC and monocytes. HeLa cell line was used as control of expression. Data from 1 representative experiment out of 3 are shown.</p

IFN-α-driven Blimp-1 expression is under the control of miR-23a and miR-125b.

<p><b>A</b>. Blimp-1α protein reduction upon transfection of miR-23a and miR-125b in HeLa cells, measured by western blot. As a control, HeLa cells transfected with empty plasmid or unrelated miRNAs (unrelated1: miR-1, unrelated2: miR-159sense) were used. Intensities of bands were measured and numbers indicate the values expressed as fold induction with respect to Gapdh. 1 representative experiment out of 3 is shown. <b>B</b>. Blimp-1 expression in HeLa cells transfected or not with single or combined miR-23a and miR-125b and treated for 24 or 48 hours with the indicated doses of IFN-α. HeLa cells transfected with empty plasmid were used as a control. Numbers indicate Blimp-1α isoform quantification expressed as fold induction with respect to Gapdh. 1 representative experiment out of 3 is shown.</p