RNase T1 mapping of IRES domain III–IV in the presence of aptamer 3-07

<p><b>Copyright information:</b></p><p>Taken from "A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III–IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId"</p><p>Nucleic Acids Research 2005;33(2):683-692.</p><p>Published online 28 Jan 2005</p><p>PMCID:PMC548359.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> 5′ end-labeled IRES RNA was digested with RNase U2, RNase T1 and an alkaline condition, respectively, were used as markers (lanes 2, 3 and 4). Cleavage reactions for markers were carried out under optimum buffer conditions for respective RNases. RNase T1 digestion with selection buffer was performed on 5′ end-labeled IRES (lane 5), the aptamer indicated (lanes 6 and 9) or mutants of aptamer 3-07 (lanes 7 and 8). Reactions were stopped and run on an 8% denaturing PAGE. Asterisks represent G triplets of domain IIId. Note that cleavage of G triplets was only protected in the presence of aptamer 3-07

Inhibition analysis of IRES-mediated translation by aptamers

<p><b>Copyright information:</b></p><p>Taken from "A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III–IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId"</p><p>Nucleic Acids Research 2005;33(2):683-692.</p><p>Published online 28 Jan 2005</p><p>PMCID:PMC548359.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Luciferase activity in the absence of an aptamer was used as a control (100%). Values represent results from at least three independent experiments. , and are equivalent to the groups in

Downregulation of <i>miR-376c</i> expression levels in bile duct carcinoma cell lines, and proteomic analysis of <i>miR-376c</i>-overexpressing HuCCT1.

<p>(<b>A</b>) Real-time PCR assay of <i>miR-376c</i> in HIBEpiC, HuCCT1, Huh28, IHGGK, TKKK, and TFK1. Expression levels were normalized to <i>RNU6B</i>, and the expression level in HIBEpiC cells was defined as 1. The significance of differences among cells was assessed by ANOVA followed by Tukey's test (*<i>P</i><0.05). (<b>B</b>) Representative 2D-DIGE images of <i>miR-376c</i>-overexpressing HuCCT1 cells. Cells were harvested 72 h after the initiation of transfection of Pre-miR-376c or the Pre-miR-negative control, and subjected to proteomic analysis. A spot downregulated by treatment with Pre-miR-376c is indicated by the arrow, which was later shown by mass spectrometry to be GRB2. (<b>C</b>) Quantitative analysis of the fluorescence intensity of the GRB2 protein spot shown in <b>B</b> (peak outlined in red).</p

<i>MiR-376c</i> Down-Regulation Accelerates EGF-Dependent Migration by Targeting <i>GRB2</i> in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line

<div><p>MicroRNA <i>miR-376c</i> was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of <i>miR-376c</i> in HuCCT1 cells is unknown. We hypothesized that <i>miR-376c</i> could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of <i>miR-376c</i>, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of <i>miR-376c</i>-overexpressing HuCCT1 cells to identify candidate targets of <i>miR-376c</i>, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to <i>miR-376c</i> overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the <i>miR-376c</i>-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the <i>miR-376c</i> gene in these cells. Proteomic analysis and subsequent validation assays showed that <i>growth factor receptor-bound protein 2</i> (<i>GRB2</i>) was a direct target of <i>miR-376c</i>. The transwell migration assay revealed that <i>miR-376c</i> significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the <i>miR-376c</i> gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of <i>miR-376c</i> in HuCCT1 cells. We revealed that epigenetic repression of <i>miR-376c</i> accelerated EGF-dependent cell migration through its target <i>GRB2</i> in HuCCT1 cells. These findings suggest that <i>miR-376c</i> functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.</p></div

Network analysis relevant to GRB2-mediated HuCCT1 migration.

<p>(<b>A</b>) Venn diagrams showing the significantly different mRNA expression levels of Pre-miR-376c and siGRB2-2 transfectants of HuCCT1 relative to appropriate controls. Expression profiles of mRNAs affected by Pre-miR-376c and siGRB2-2 in EGF-treated HuCCT1 cells were conducted by microarray analysis. The numbers of genes regulated by Pre-miR-376c and siGRB2-2 are indicated. (<b>B</b>) The network of the identified molecules regulated by both Pre-miR-376c and siGRB2-2 in this study were connected with EGF, EGFR and GRB2 by IPA analysis. Numbers below the upregulation (red) and downregulation (green) symbols represent the fold changes by Pre-miR-376c treatment; numbers in parentheses represent the fold changes by siGRB2-2 treatment. Solid and dotted lines indicate direct and indirect gene relationships, respectively. (<b>C and D</b>) Real-time PCR analysis of <i>IL1B</i> (<b>C</b>) and <i>MMP9</i> (<b>D</b>) expression levels in EGF-treated HuCCT1 cells after transfection with Pre-miR-376c and siGRB2-2. Pre-miR-negative control and nonspecific non-silencing siRNA were used as negative controls (NC). For quantitative comparisons, expression levels were normalized to <i>GAPDH</i>. The expression levels in negative controls were set to 1.0. The significance of differences between means was determined by Student's <i>t</i>-test. * <i>p</i><0.05.</p

Validation of <i>GRB2</i> as a <i>miR-376c</i> Target.

<p>(<b>A</b>) Western blotting analysis of GRB2 protein levels in HuCCT1 cells transfected with Pre-miR-376c or the Pre-miR negative control (NC). ACTB was monitored as an internal control. Relative GRB2 expression levels were calculated and are indicated below the bands. (<b>B</b>) <i>GRB2</i> mRNA expression levels in HuCCT1 cells transfected with Pre-miR-376c or the Pre-miR negative control (NC). The <i>GRB2</i> expression level was normalized to <i>GAPDH</i>. The expression level of the NC sample was defined as 1. The significance of differences between means was determined by Student's <i>t</i>-test. (<b>C</b>) The sequences of the mature <i>miR-376c</i> and its putative target site in the 3'-UTR of <i>GRB2</i>. The target site corresponding to the seed sequence of <i>miR-376c</i> was converted via mutation; the mutation introduced into the <i>miR-376c</i> recognition site of <i>GRB2</i> 3'-UTR in the reporter plasmid is also shown. (<b>D</b>) <i>GRB2</i> 3'-UTR luciferase reporter assay. Reporter vector (pMIR-GRB2 [GRB2] or pMIR-GRB2mt [GRB2mt]) and Pre-miR molecule (Pre-miR-376c [376c] or Pre-miR negative control [NC]) were co-transfected into HuCCT1 cells. Renilla luciferase vector pRL-TK was used as an internal control. Luciferase expression levels of Pre-miR negative control (NC) were set to 1.0. The significance of differences between means was determined by Student's <i>t</i>-test.</p

<i>MiR-376c</i> represses cell migration via <i>GRB2</i> reduction.

<p>(<b>A</b>) Transwell migration assay of HuCCT1 cells transfected with Pre-miR-376c. Medium containing 5 ng/ml EGF in the lower chamber served as a chemoattractant. After 24 h of transfection, migrating cells were stained and counted. Data are presented as the ratio of the number of migrating Pre-miR-376c (376c)-transfected cells to that of cells transfected with the Pre-miR-negative control (NC), in the presence or absence of EGF. Cell migration of NC in the presence of EGF was set to 1.0. The significance of differences among treatments was assessed by ANOVA followed by Tukey's test (* <i>p</i><0.05). (<b>B</b>) Western blotting analysis of the GRB2 protein level in HuCCT1 cells transfected with the siRNAs. Two siRNA molecules targeting <i>GRB2</i> (siGRB2-1 and siGRB2-2) and negative control siRNA (NC) were used. ACTB was used as an internal control. (<b>C</b>) Real-time PCR analysis of the <i>GRB2</i> mRNA level in HuCCT1 cells transfected with the siRNAs. The expression level of cells transfected with negative control siRNA (NC) was set to 1.0. The <i>GRB2</i> expression levels were normalized to <i>GAPDH</i>. (<b>D</b>) Transwell migration assay of HuCCT1 cells transfected with the siRNAs. Data are presented as numbers of migrating siRNA-transfected cells relative to cells transfected with the negative control siRNA (NC), in the presence or absence of EGF. Migration of the negative controls in the presence of EGF was set to 1.0. The significance of differences among treatments was assessed by ANOVA followed by Tukey's test (* <i>p</i><0.05).</p

Methylation of <i>miR-376c</i>.

<p>(<b>A</b>) Location of the six CpG sites (sites I–VI) upstream of <i>miR-376c</i>, in the putative promoter region of the gene. (<b>B</b>) Bisulfite sequencing analysis of these CpG sites in HIBEpiC and HuCCT1. The unmethylated levels of the six sites were expressed as percentages of methylation reference values. A mutation in the genome sequence of HuCCT1 was found at CpG site III. (<b>C</b>) Real-time PCR analysis of <i>miR-376c</i> expression levels in HuCCT1 cells treated with the DNA-demethylating agent 5-AZA-dCR and/or the HDAC inhibitor TSA. After treatment with 10 µM of 5-AZA-dCR for 3 days, HuCCT1 cells were incubated with TSA (0.1, 0.5, or 1.0 µM) for a further 24 h. Expression levels were normalized to <i>RNU6B</i>. The expression level in untreated HuCCT1 cells was defined as 1 (lane 1). Differences among treatments were tested by ANOVA followed by Tukey's test (* <i>p</i><0.05).</p

Proteins downregulated by <i>miR-376c</i> overexpression in HuCCT1 cells in proteomic analysis.^*

*<p>The protein spots that had significant differences in intensity between the control- and Pre-miR-376c-transfected HuCCT1 cells (down-regulation by more than 1.2-fold change, <i>p</i>≤0.011) are listed in proteomic analysis. FC: fold change. The GRB2 protein predicted by TargetScan as a target of <i>miR-376c</i> is shown in bold.</p