15 research outputs found

    <i>Chop</i> mRNA is a direct target of miR-615-3p.

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    <p>A region of the <i>Chop</i> 3′UTR containing the putative miR-615-3p binding site was cloned into the pMIR-report vector downstream of the luciferase coding region (p-MIR-Chop). The 3′UTR segment with the putative miR-615-3p binding site mutated (p-MIR-Chop-mut) was also cloned into the pMIR-report vector. HEK293 cells were co-transfected with the respective reporter plasmid and precursor of miR-615-3p (pre-miR-615-3p) or a negative control precursor molecule. Relative luciferase activity (normalized to renilla) was measured 24 hours after the transfection. Data are expressed relative to the wild-type binding site transfected cells treated with a negative control precursor molecule and (n = 5 independent experiments), * P<0.05.</p

    miR-106b-responsive gene landscape identifies regulation of Kruppel-like factor family

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    <p>MicroRNA dysregulation is a common feature of cancer and due to the promiscuity of microRNA binding this can result in a wide array of genes whose expression is altered. miR-106b is an oncomiR overexpressed in cholangiocarcinoma and its upregulation in this and other cancers often leads to repression of anti-tumorigenic targets. The goal of this study was to identify the miR-106b-regulated gene landscape in cholangiocarcinoma cells using a genome-wide, unbiased mRNA analysis. Through RNA-Seq we found 112 mRNAs significantly repressed by miR-106b. The majority of these genes contain the specific miR-106b seed-binding site. We have validated 11 genes from this set at the mRNA level and demonstrated regulation by miR-106b of 7 proteins. Combined analysis of our miR-106b-regulated mRNA data set plus published reports indicate that miR-106b binding is anchored by G:C pairing in and near the seed. Novel targets Kruppel-like factor 2 (KLF2) and KLF6 were verified both at the mRNA and at the protein level. Further investigation showed regulation of four other KLF family members by miR-106b. We have discovered coordinated repression of multiple members of the KLF family by miR-106b that may play a role in cholangiocarcinoma tumor biology.</p

    Embelin induced altered nuclear morphology in cholangiocarcinoma cell lines.

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    <p>(A) KMCH cells were treated for 24 hours with TRAIL at the indicated concentrations with or without embelin (1 and 10 µM). Cells were then stained with DAPI and bright nuclei were counted as a percentage of total nuclei. Data from one experiment are plotted as percent DAPI-positive nuclei on the vertical axis. (B) Mz-ChA-1 cells were treated with TRAIL (4 ng/mL) or medium for 24 hours with 5 or 10 µM embelin, and DAPI-positive nuclei counted as a percent of total cells. Data are mean of 3 experiments +/− standard error of the mean. n.s. = not significantly different. * p<0.05, ** p<0.01 by ANOVA with Bonferroni compared to medium alone. (C) Rat BDEneu cholangiocarcinoma cells were treated with DMSO (Vehicle; open bar) or embelin (50 µM, filled bar) for 48 hours, followed by DAPI staining. Data are mean of 3 experiments +/− SEM. *** p<0.001 compared to vehicle, Students <i>t</i>-test. (D) Vehicle-treated Mz-ChA-1 cells were stained with DAPI and imaged by epifluorescence without fixation. Healthy nuclei (indicated by grey outlines) did not stain with DAPI while a sporadic apoptotic nucleus (arrow) was brightly stained. Bar = 10 µm. (E) DAPI-positive nuclei of Mz-ChA-1 cells treated with embelin (15 µM for 24 hours) did not show characteristic apoptotic fragmentation or pyknosis. (F) Mz-ChA-1 cells were treated with DMSO (Veh), embelin (15 µM), or staurosporine followed by analysis of DNA fragmentation on a 2% agaraose gel. Vehicle treatmetn was for 24 hours. Embelin and staurosporine treatments were for 4, 8, 16, and 24 hours. M = 100 bp DNA marker. The gel was stained with ethidium and photographed and the image was inverted to show DNA as a dark signal on a light background. Images in Panel D, E, and F were adjusted for brightness and contrast to ensure that features were visible and the entire image was treated equally.</p

    Ceramide stimulated the binding of STAT3, but not NF-κB subunit P65 or c-Jun, to <i>HAMP</i> promoter.

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    <p>(<b>A</b>) Chromatin, isolated from HepG2 cells treated with 60 μM C2 ceramide or solvent (solv.) for 8 hours and fixed with 1% formaldehyde, was incubated with antibodies specific for STAT3, NF-κB subunit P65 or c-Jun for chromatin immunoprecipitation (ChIP) assays, as described in the experimental procedures. Normal rabbit or mouse IgG were employed as negative controls. Immunoprecipitated and purified chromatin and total input chromatin (loading control) were used for PCR to amplify a <i>HAMP</i> promoter region harboring corresponding consensus sequences by using specific primers. Representative images of amplicons, analyzed by DNA agarose gel electrophoresis, visualized by ethidium bromide staining, and captured using Gel Doc XR+ system (Bio-Rad), are shown. (<b>B</b>) ChIP assays were performed with HepG2 cells treated with recombinant IL-6 or PBS (control) by using anti-STAT3 antibodies, as described above.</p
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