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

<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

Ceramide did not induce ER stress in HepG2 cells (A)

<p>HepG2 cells, treated with 60 μM C2 ceramide and solvent (solv.) control or 10 μg/ml tunicamycin (TUNI), as positive control, for 8 hours were used for XBP1 splicing assay, as described in the experimental procedures. <b>(B)</b> Whole cell lysates from solvent (solv.) or ceramide-treated HepG2 cells were employed for western blotting to determine GRP78 and CHOP protein expression using specific antibodies. An anti-gapdh antibody was employed as a protein loading control.</p

Embelin induced altered nuclear morphology in cholangiocarcinoma cell lines.

<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-induced <i>HAMP</i> up-regulation was not dependent on c-Jun N-terminal kinase (JNK) phosphorylation.

<p>HepG2 cells treated with ceramide or solvent in the presence of 50 μM chemical JNK inhibitor, Sp600125 or DMSO (control) were used to isolate protein (<b>A</b>) for western blotting to determine phosphorylated (P-JNK) and total JNK protein (T-JNK) levels using specific antibodies or to isolate RNA (<b>B</b>) to determine <i>HAMP</i> mRNA expression by Taqman qPCR. <i>HAMP</i> expression in ceramide-treated cells was calculated as fold expression of that in respective control cells treated with solvent. Asterisks indicate statistical significance (P<0.05).</p

Inhibition of proliferation and cell cycle arrest by embelin.

<p>(A) Cell proliferation was measured by MTT and cell number measured by absorbance at 540 nm (Abs 540 nm). Signal represents the mean (n = 4) +/− standard error of the mean, normalized to the starting value (day 0, set at 100%). Cells treated with embelin (15 µM) are plotted with a solid line and filled symbols and vehicle-treated cells are plotted with a dashed line and open symbols. ** p<0.01 and *** p<0.001 versus vehicle at the same time point, ANOVA with Bonferroni correction. Values for HuCCT were not significantly different at any time point. (B) Cell cycle analysis of Mz-ChA-1 cells was performed by propidium iodide staining followed by flow cytometry. A histogram of propidium iodide stained cells is shown for DMSO-treated and embelin-treated cells (15 µM, 24 hours). (C) Quantitation of the percentage of cells with 2N or 4N nuclear DNA content, and cells that are in S phase (DNA content intermediate between 2N and 4N). Representative experiment of 3 independent treatments.</p

The effect of ceramide on the phosphorylation of STAT3, JNK and NF-κB in HepG2 cells.

<p>Protein lysates isolated from HepG2 cells treated with 60 μM C2 ceramide or solvent control, and 40 ng/ ml recombinant IL-6 or PBS control for 8 hours, were employed for western blotting to determine the level of total or phosphorylated proteins. Total STAT3 (T-STAT3) or STAT3 phosphorylated on tyrosine 705 (P-STAT3) (<b>A</b>), total (T-P65) or phosphorylated (P-P65) NF-κB subunit P65, and total (T-JNK) or phosphorylated-JNK (P-JNK) (<b>B</b>) protein levels were determined by using specific antibodies. An anti-gapdh antibody was employed as a protein loading control.</p

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

<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

Embelin partially inhibited caspase activation and did not induce caspase-dependent cell death in cholangiocarcinoma cells.

<p>(A) Mz-ChA-1 cells were treated with embelin for 24 hours and caspase 3/7 activity measured biochemically. Untreated cells were used for comparison and caspase activity in untreated cells normalized at 1.0. (B) BDEneu cells were treated with embelin (48 hours) and caspase 3/7 activity measured. (C) Caspase 3/7 activity was measured at an earlier time point (4 hours) in Mz-ChA-1, KMCH, and HuCCT cells to test for early caspase activation. Following 4 hours of vehicle, embelin (15 µM) or staurosporine (1 µg/mL), caspase 3/7 activity was measured biochemically. (D) BDEneu cells treated with 50 µM embelin for 48 hours were assayed for DAPI-positive nuclei with and without co-treatment with the caspase inhibitor Z-VAD-fmk (50 µM). DAPI-positive nuclei are presented as percent of total cells, n = 3, mean +/− SEM. Comparison of embelin versus embelin+Z-VAD-fmk was not significantly different. Panels A, B, C & D data are mean of 3 or 4 experiments +/− SEM; ** p<0.01, *** p<0.001 versus vehicle, ANOVA with Bonferroni correction. (E) Mz-ChA-1 cells were treated with embelin (5–15 µM) in DMSO or DMSO alone (Veh) for 24 hours. Whole cell lysates were blotted for PARP. Actin was included as a loading control. Apparent molecular weight for each protein is indicated to the right.</p

Ceramide analogs induced <i>HAMP</i> transcription.

<p>(<b>A</b>) <i>HAMP</i> mRNA expression in HepG2 cells, treated with 30 μM or 60 μM of C2 and C6 ceramide or solvent (solv.) control for 8 hours, was determined by Taqman qPCR, as described in experimental procedures. <i>HAMP</i> mRNA levels in ceramide-treated cells was expressed as fold change of that in solvent-treated cells. (<b>B</b>) HepG2 cells were treated with 60 μM C2 ceramide or solvent in the presence of either 1μg/ml actinomycin D (ACTD) or DMSO as control. <i>HAMP</i> mRNA expression, determined by qPCR, in treated cells was calculated as fold change of that in control cells, incubated with solvent and DMSO. (<b>C</b>) The effect of ACTD on <i>HAMP</i> up-regulation induced by ceramide was shown by calculating <i>HAMP</i> expression in C2 ceramide-treated cells as fold change of that in cells treated with solvent. (<b>D</b>) HepG2 cells, transfected with pGL-3 basic vector harboring 0.6kbp <i>HAMP</i> promoter (<i>HAMP</i>-Prom-Luc) or empty pGL-3 basic vector (vector), and pRL-SV40 plasmid as control for transfection efficiency, were treated with 60 μM C2 ceramide or solvent for 8 hours. Dual luciferase report assays were performed, as described in the experimental procedures. <i>HAMP</i> promoter activity, expressed in relative luciferase units, was calculated as fold change of that in cells transfected with the empty vector and treated with solvent. Asterisks indicate statistical significance (P<0.05).</p

The effect of STAT3 response element mutation on <i>HAMP</i> promoter activation by ceramide.

<p>The single STAT3 response element within 0.6kbp <i>HAMP</i> promoter region cloned in pGL3 basic vector was mutated, as described in the experimental procedures. HepG2 cells, transfected with pGL3-basic vector harboring wild-type (WT) or mutated (ΔSTAT3) 0.6 kb <i>HAMP</i> promoter, or empty vector (vector) as negative control, and pRL-SV40 plasmid, as control for transfection efficiency, were treated with (<b>A</b>) 60 μM C2 ceramide (C2) and solvent control (solv.) or (<b>B</b>) 40 ng/ ml IL-6 and PBS control for 8 hours. Dual luciferase reporter assays were performed, as described in the experimental procedures. <i>HAMP</i> promoter activity, expressed in relative luciferase units, in treated cells was calculated as fold change of that in control cells transfected with empty vector-and treated with solvent. Asterisks indicate statistical significance (P<0.05).</p