26 research outputs found

    Additional file 2: Table S1. of Gene expression profiling analysis of CRTC1-MAML2 fusion oncogene-induced transcriptional program in human mucoepidermoid carcinoma cells

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    A list of CRTC1-MAML2 fusion-regulated genes in fusion-positive H3118 MEC cells. Gene expression profiling analyses were performed on fusion/MAML2-knockdown H3118 cells and MAML2-knockdown HSY cells in comparison with their corresponding control cells. Differentially expressed genes with absolute fold change > = 2 and p < 0.05 were identified. The differentially expressed genes in fusion/MAML2 knockdown H3118 cells showing the same regulated direction in MAML2 knockdown HSY cells were filtered out. The “positive” and “negative” signs denote upregulated or down-regulated genes in KD compared to control groups, respectively. The asterisk indicates the common gene in different regulatory direction between HSY and H3118. Table S2. A list of differentially regulated genes affected by CREB depletion in human fusion-positive MEC, but not fusion-negative cells. The differentially expressed genes after CREB was depleted were identified in both fusion-positive MEC H3118 cells and fusion-negative HSY cells. Those CREB-regulated genes in H3118 cells showing the same regulated direction in fusion-negative HSY were then filtered out. The “positive” and “negative” signs denote up-regulated or down-regulated genes in KD compared to control groups, respectively. The asterisk indicates the common gene in different regulatory direction between HSY and H3118 cells. Table S3. A list of common differentially expressed genes affected by CRTC1-MAML2- or CREB-depletion in human MEC H3118 cells. This gene list represents candidate genes regulated by fusion/CREB interaction. The “positive” and “negative” signs denote up-regulated or down-regulated genes in KD compared to control groups, respectively. The asterisk indicates the common gene in different regulatory direction between fusion and CREB. (PDF 707 kb

    Additional file 1: Figure S1. of Gene expression profiling analysis of CRTC1-MAML2 fusion oncogene-induced transcriptional program in human mucoepidermoid carcinoma cells

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    Volcano plots showed differential expression between MAML2 TAD shRNA and control groups in fusion-negative HSY cells (A) and in fusion-positive H3118 cells (B). The vertical lines corresponded to 2.0-fold up and down, respectively, and the horizontal line represented a p-value of 0.05. NC denoted no change. “Down” or “Up” referred to significantly down-regulated or upregulated genes. Several fusion-regulated genes in H3118 cells were indicated. Figure S2. Validation of a subset of CRTC1-MAML2 fusion-regulated genes in human MEC H292 cell line. Real-time RT-PCR assays were performed in H292 cells that were depleted of fusion/MAML2 or MAML2 only for a subset of CRTC1-MAML2 fusion candidate genes identified in human MEC H3118 cells. Figure S3. Volcano plots showed differential expression between CREB shRNA and control groups in fusion-negative HSY cells (A) and in fusion-positive H3118 cells (B). The vertical lines corresponded to 2.0-fold up and down, respectively, and the horizontal line represented a p-value of 0.05. NC denoted no change. “Down” or “Up” referred to significantly down-regulated or up-regulated genes. (PDF 4339 kb

    <i>miR-34</i> Modulates Innate Immunity and Ecdysone Signaling in <i>Drosophila</i>

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    <div><p>microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. Here we show that the conserved miRNA <i>miR-34</i> regulates innate immunity and ecdysone signaling in <i>Drosophila</i>. <i>miR-34</i> over-expression activates antibacterial innate immunity signaling both in cultured cells and <i>in vivo</i>, and flies over-expressing <i>miR-34</i> display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas <i>miR-34</i> knockout animals are defective in antibacterial defense. In particular, <i>miR-34</i> achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes <i>Dlg1</i> and <i>Eip75B</i>. In addition, our study reveals a mutual repression between <i>miR-34</i> expression and ecdysone signaling, and identifies <i>miR-34</i> as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, we identify <i>cis</i>-regulatory genomic elements and <i>trans</i>-acting transcription factors required for optimal ecdysone-mediated repression of <i>miR-34</i>. Taken together, our study enriches the repertoire of immune-modulating miRNAs in animals, and provides new insights into the interplay between steroid hormone signaling and innate immunity.</p></div

    Over-expression of <i>miR-34</i> activates innate immunity signaling.

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    <p>(<b>A</b>) Total RNA was isolated from male progeny from crossing flies carrying the ubiquitously expressed <i>da-Gal4</i> driver and a temperature-sensitive <i>Gal80</i> transgene (<i>da>Gal4 tub-Gal80</i><sup><i>ts</i></sup>) to UAS-shRNA lines targeting <i>Drosha</i> or the control <i>gfp</i>. Flies crosses were kept at 18°C and progeny were shifted to 29°C for 5 days upon eclosure to induce shRNA expression. Steady-state levels of mRNAs encoding Drosha and several primary miRNA transcripts were measured by qRT-PCR, and normalized to levels of the <i>RpL32</i> mRNA. RNA isolated from <i>da>gfp shRNA</i> males serves as negative control. (n = 3). (<b>B</b>) Flies were left untreated (- <i>E</i>. <i>coli</i>) or infected by <i>E</i>. <i>coli</i> via septic injury (+ <i>E</i>. <i>coli</i>), total RNAs were extracted 6 hours post-infection and mRNAs encoding the AMP Diptericin was measured and normalized to levels of <i>RpL32</i> (n = 5; mean + standard deviation (<b>SD</b>)). (<b>C-E</b>) Select miRNAs were over-expressed in flies by crossing UAS-miRNA transgenic lines <i>da>Gal4 tub-Gal80</i><sup><i>ts</i></sup> flies. Flies crosses were kept at 18°C and progeny were shifted to 29°C for 5 days upon eclosure to induce miRNA expression. (<b>C</b>) Northern blot shows levels of select miRNAs (right) in control and miRNA over-expression flies. 2S rRNA serves as loading control. In addition, flies were either uninfected (<b>D</b>) or infected with <i>E</i>. <i>coli</i> via septic injury (<b>E</b>). Total RNA was isolated from flies 6 hrs post-infection and levels of <i>Diptericin</i> mRNA were measured by RT-qPCR and normalized to the <i>RpL32</i> mRNA. RNA samples from <i>da>gfp shRNA</i> flies serves as control. Note that levels of the <i>Diptericin</i> mRNA in non-infected and <i>E</i>. <i>coli</i>-infected <i>da>gfp shRNA</i> flies serve as baseline controls in both <b>D</b> and <b>E</b> (n≥4). (<b>F</b>) A Northern blot shows <i>miR-34</i> expression levels in naïve S2 cells and <i>miR-34</i> overexpression cells (both were treated with 20-HE at 1 μM for 24 hrs). (<b>G</b>) S2 cells over-expressing <i>miR-34</i> and control cells were both treated with 20 hydroxy-ecdysone (<b>20-HE</b>) at 1 μM for 24 hrs. Subsequently cells were either left untreated or treated for 6 hrs with a crude lipopolysaccharide sample at 10 μg/mL, which contains the immune stimulator <i>p</i>eptido<i>g</i>lyca<i>n</i> (<b>PGN</b>). Total RNA was isolated and levels of <i>Diptericin</i> mRNA were measured by RT-qPCR and normalized to the <i>RpL32</i> mRNA (n = 3). (<b>H</b>) Canonical components of IMD signaling were depleted in <i>miR-34</i> over-expressing cells using dsRNAs targeting IMD pathway components (below) or a control dsRNA against the firefly luciferase gene. Cells were first treated with 20-HE for 24 hours, and subsequently were either left untreated or treated with PGN, and levels of <i>Diptericin</i> mRNA were measured by RT-qPCR and normalized to the <i>RpL32</i> mRNA (n = 3). (<b>I</b>) <i>UAS-miR-34</i> or the control <i>UAS-sh-gfp</i> flies were crossed to <i>da>Gal4 tub-Gal80</i><sup><i>ts</i></sup> flies. Flies crosses were kept at 18°C. Upon eclosure, progeny of appropriate genotypes were collected and shifted to 29°C for 7 days. Flies in groups of 45 were subsequently injected with a concentrated culture of <i>Erwinia carotovora carotovora 15</i> (<i>Ecc15</i>) or PBS (non-infection control) and kept at 29°C. Fly survival was recorded daily up to day 8 post-infection and plotted (n≥3; <i>p</i><0.05 between <i>Ecc15</i>-infected control and <i>miR-34</i><sup><i>OX</i></sup> (<i>da>miR-34 Gal80</i><sup><i>ts</i></sup>) flies). (<b>J-K</b>) Control or <i>miR-34</i><sup><i>OX</i></sup> flies were infected by injecting a concentrated culture of <i>Ecc15</i> (<b>J</b>) or overnight culture of <i>Enterobacter cloacae</i> (<b>K</b>). At various time points post-infection, groups of 3 flies in <b>J</b> (and groups of 4 flies in <b>K</b>) were collected and homogenized in sterile PBS. Fly homogenates were diluted and plated onto Ampicillin- (in <b>J</b>) or Nalidixic acid-containing (in <b>K</b>) LB plates, and the resultant colonies were counted one day later. Shown are <i>c</i>olony-forming <i>u</i>nits (<b>CFU</b>s) per fly (n≥4).</p

    <i>Eip75B</i> is another <i>miR-34</i> target gene that modulates innate immunity signaling.

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    <p>(<b>A-B</b>) S2 cells over-expressing <i>miR-34</i> or control cells were either left untreated or treated with 20-HE (labeled on top). Cell lysates were subject to immunoblot using various antibodies against Eip74EF, Eip75B, BrC or the control Tubulin (<b>A</b>). Levels of the indicated proteins in 20-HE-treated cell samples were quantified in <b>B</b> (n≥3). (<b>C</b>) Reporter constructs were generated that carry either a wildtype (WT) or mutant (mut) <i>miR-34</i> binding site derived from the <i>Eip75B</i> ORF. Seed region of <i>miR-34</i> was highlighted in green. (<b>D</b>) The reporter constructs were transfected into S2 cells together with or without a <i>miR-34</i> expression construct, and reporter activities were measured (n = 3). (<b>E-F</b>) S2 cells treated with dsRNA against <i>Eip75B</i> or a control dsRNA were first treated with 20-HE, and subsequently were either left untreated or treated with PGN. Total RNA was isolated and levels of the <i>Diptericin</i> (<b>E</b>) and <i>Eip75B</i> mRNA (<b>F</b>) were measured and normalized to <i>RpL32</i> (n≥3). (<b>G</b>) Various combinations of <i>miR-34</i> and <i>Eip75B</i> expression constructs were transfected into S2 cells. Cells were treated with 20-HE for 24 hrs, and total RNA was isolated and levels of the <i>Diptericin</i> mRNA were measured and normalized to the <i>RpL32</i> control mRNA (n = 3).</p

    Mapping <i>cis</i>-regulatory elements required for ecdysone-mediated repression of <i>miR-34</i>.

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    <p>(<b>A</b>) The <i>miR-34</i> locus contains five candidate regulatory regions (<b><i>P1</i> through <i>P5</i></b>) that are repressed in response to ecdysone treatment. (<b>B-C</b>) S2 cells were treated with ecdysone for various times (below) and levels of various mRNAs (<b>B</b>) or pri-miRNAs (<b>C</b>) were measured and normalized to the control <i>RpL32</i> mRNA (n≥3). (<b>D</b>) DNA fragments derived from various regulatory regions were placed upstream of a luciferase reporter gene. S2 cells transfected with these reporter constructs were left untreated or treated with ecdysone (20-HE) and reporter activities were measured. Fold change in ecdysone-mediated repression of reporter activity is shown (n≥3; mean + SD). Cells transfected with a firefly luciferase reporter gene driven by the regulatory region derived from the <i>traffic jam</i> gene, which is not responsive to ecdysone treatment, serve as control. (<b>E</b>) A schematic of the <i>P2</i> region. Predicted BrC-binding sites are shown as shaded boxes, whereas open and filled boxes represent one of the three predicted Twi- and Srp-binding sites, respectively. F1-F5 represent various fragments tested for BrC occupancy in <b>D</b>. In addition, various truncated fragments tested in reporter assays in <b>G</b> are shown on the left. (<b>F</b>) Chromatin immunoprecipitation assay was employed in cultured S2 cells to measure BrC occupancy in various regions of <i>P2</i> (schematic shown in <b>E</b>, n = 3; mean + SD). (<b>G</b>) Reporter constructs containing either full length or various truncated <i>P2</i> fragments (in <b>E</b>) were transfected into S2 cells. Cells were left untreated or treated with ecdysone (20-HE) and reporter activities were measured. Ecdysone-mediated repression of reporter activity is shown (n≥3; mean + SD).</p

    Identification of trans-acting transcription factors required for ecdysone-mediated repression of <i>miR-34</i>.

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    <p>(<b>A-F</b>) S2 cells transfected with various dsRNAs were left untreated or treated with ecdysone (20-HE) at 1 μM for 48 hrs. Total RNA was isolated and levels of mature <i>miR-34</i> were measured by Northern blot (<b>A</b>) and normalized to the 2S rRNA (<b>B</b>; n = 3). In addition, levels of the primary <i>miR-34</i> transcript (<b>C</b>), or the mRNAs for <i>BrC</i> (<b>D</b>) or <i>EcR</i> (<b>E</b>) were measured and normalized to the control <i>RpL32</i> mRNA (n = 3; mean + SD). In addition, levels of the BrC protein were measured by immunoblot (<b>F</b>). Note that multiple isoforms of the BrC protein are expressed in S2 cells upon ecdysone treatment and are responsive to dsRNA-mediated knockdown. (<b>G</b>) RNA was extracted from third instar <i>BrC</i><sup><i>npr6</i></sup> mutant or wildtype (WT) larvae and levels of <i>pri-miR-34</i> were measured and normalized to the control <i>RpL32</i> mRNA (n = 4). (<b>H</b>) <i>BrC</i><sup><i>npr6</i></sup> mutant and wildtype larvae was either left untreated or infected by a concentrated culture of <i>E</i>. <i>coli</i> via septic injury. Total RNA was isolated 6 hrs post-infection and levels of <i>Dpt</i> mRNA were measured and normalized to the control <i>RpL32</i> mRNA (n = 4). (<b>I</b>) S2 cells transfected with dsRNAs targeting various transcription factors (below). These cells were left untreated or treated with ecdysone, and levels of <i>pri-miR-34</i> were measured and normalized to the control <i>RpL32</i> mRNA (n≥3; mean + SD).</p

    <i>miR-34</i> deficiency compromises innate immunity.

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    <p>(<b>A</b>) A Northern blot shows levels of <i>miR-34</i> and the control <i>2S</i> rRNA in <i>miR-34</i> knockout flies (<b><i>KO</i></b>) or knockout flies carrying a <i>miR-34</i> rescue transgene (control, ctr). (<b>B</b>) Flies were either uninfected or infected with <i>E</i>. <i>coli</i> via septic injury. Total RNA was isolated and levels of the <i>Diptericin</i> mRNA were measured by RT-qPCR and normalized to the <i>RpL32</i> control (mean + SD; n = 3). (<b>C-D</b>) <i>miR-34</i><sup><i>KO</i></sup> and control flies were reared in standard food supplemented with antibiotics. Age-matched fly progeny (young– 3d, old– 22d) were either uninfected (<b>C</b>) or infected with <i>E</i>. <i>coli</i> via septic injury (<b>D</b>), and levels of the <i>Diptericin</i> mRNA were measured by RT-qPCR and normalized to the <i>RpL32</i> control (n≥3). In both panels, the <i>Dpt</i>/<i>RpL32</i> ratio in non-infected 3d old control flies serves as baseline. (<b>E-F</b>) Groups of 45 age-matched <i>miR-34</i><sup><i>KO</i></sup> and control flies (<b>E</b>, young– 3d; <b>F</b>, old– 23d) were injected with a concentrated culture of <i>Ecc15</i> or PBS. Fly survival was recorded daily and plotted (n≥3; <i>p</i><0.001 between <i>Ecc15</i>-infected control and <i>miR-34</i><sup><i>KO</i></sup> flies). (<b>G-H</b>) A similar group of age-matched control or <i>miR-34</i><sup><i>KO</i></sup> flies (as in <b>E</b> and <b>F</b>) were infected by injecting a concentrated culture of <i>Ecc15</i>. At various time points post-infection, groups of 3 flies were collected and homogenized in sterile PBS. Note that due to lethality, 1 fly per group was used for a subset of data points in <i>miR-34</i><sup><i>KO</i></sup> flies 2 days post-infection. Fly homogenates were diluted and plated onto Ampicillin-containing LB plates, and the resultant colonies were counted one day later. Shown are <i>c</i>olony-forming <i>u</i>nits (<b>CFU</b>s) per fly (n≥5). (<b>I</b>) Groups of 4–7 d <i>miR-34</i><sup><i>KO</i></sup> and control flies were injected with a concentrated culture of <i>Enterobacter cloacae</i> or PBS. Fly survival was recorded daily and plotted (n≥3; <i>p</i><0.001 between <i>Enterobacter cloacae</i>-infected control and <i>miR-34</i><sup><i>KO</i></sup> flies). (<b>J</b>) A similar set of flies (as in <b>I</b>) were infected by injecting an overnight culture of <i>Enterobacter cloacae</i>. At various time points post-infection, groups of 4 flies were collected and homogenized in sterile PBS. Fly homogenates were diluted and plated onto Nalidixic acid-containing LB plates, and the resultant colonies were counted one day later. Shown are <i>c</i>olony-forming <i>u</i>nits (<b>CFU</b>s) per fly (n = 5).</p

    Tks5 expression in breast tissue and breast cancer.

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    <p>A) 293 cells were transfected with empty vector, or vectors expressing Tks4 or Tks5. Cells were pelleted, fixed, embedded in paraffin and processed for immunohistochemistry with an anti-Tks5 antibody. B) Representative images of normal breast lobules and duct, and ductal carcinoma in situ, stained with anti-Tks5 antibodies. Red arrows indicate areas with intense Tks5 staining. Scale bar represents 100μm. C) Representative images of primary invasive breast cancer specimens, at low and high magnification, to illustrate the range of Tks5 expression observed. Scale bars represent 100μm. Quantification of 163 specimens is shown on the right. D) Kaplan-Meier survival curves for patients with high (red) and low (black) Tks5α mRNA levels. E) Kaplan-Meier survival curves for stage I/II patients with high (red) and low (black) Tks5α mRNA levels. F) Kaplan-Meier survival curves for stage III/IV patients with high (red) and low (black) Tks5α mRNA levels.</p
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