10 research outputs found
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
MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer
<div><p>Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.</p> </div
Prediction of miR-200c interaction sites in MUC4 and MUC16 genes.
<p>Possible miR-200c targeting regions in MUC4 and MUC16 were identified by using the RegRNA MicroRNA target prediction web server (<a href="http://regrna.mbc.nctu.edu.tw/index1.php" target="_blank"><u>http://regrna.mbc.nctu.edu.tw/index1.php</u></a>). A, RegRNA miRNA target prediction shows that miR-200c binds between base pairs 820-842 in the first exon of MUC4. B, In MUC16 mRNA, the miR-200c is predicted to bind nine different exons including E1, E3, E19, E39, E44, E49, E54, E64 and E73. The numbers indicate the region of mRNAs that interact with miR-200c.</p
microRNA-200c targets MUC16.
<p>(A) S2.028 and (B) T3M-4-miR200c and respective vector control transfected cell lysates were separated by SDS-Agarose gel electrophoresis and subjected to western blot using an anti-MUC16 antibody (left panels). Band intensity was quantified by densitometry and analyzed using the imageJ program (right panels). Significant reductions of both high and low molecular weight MUC16 protein isoforms were observed in miR-200c expressing S2.028 (A) and T3M-4 cells (B) than compared to vector control cells. α-tubulin was used as a loading control.</p
Quantitative analysis of miR-200c expression in human pancreatic cancer cell lines and ectopic expression of miR-200c in S2.028 and T3M-4 cells.
<p>(A) The expression of miR-200c in seven pancreatic cancer cells were determined by Real-time PCR. Each sample was run in quadruplicate and error bars represent SD. S2.028 (B) and T3M-4 cells (C) stably expressing the primary transcript of miR-200c were evaluated for miR-200c expression by Real-time PCR. Each measurement was carried out in triplicate. These values were normalized with internal control U6 rRNA. The fold increase in transcript levels over vector control is expressed as Mean ± S.D. The p value was determined by using the Student’s t-test. Differences with a p value < 0.05 were considered statistically significant.</p
MUC1.CT directly interacts with transcriptional repressor ZEB1.
<p>A) Co-immunoprecipitation (Co-IP) was utilized to evaluate the interaction of the MUC1.CT and ZEB1 in S2.013.Neo and S2.013.MUC1 cells. Cell lysates were immunoprecipitated with an antibody against the MUC1.CT, and the subsequent western was blotted with an antibody to ZEB1. Non-immunoprecipitated lysates were used as a loading control, and steady state levels of ZEB1 were higher in S2.013.MUC1 cells compared to S2.013.Neo cells. B-C) Proximity ligation assay (PLA) was used to visualize MUC1.CT and ZEB1 interaction in S2.013.Neo and S2.013.MUC1 cells, B, and Panc1.Neo and Panc1.MUC1 cells, C (with representative compressed z-stack image for each). These data show quantitative levels of MUC1.CT and ZEB1 interactions, indicating that MUC1.CT and ZEB1 interact in both cell lines. In the S2.013.Neo and S2.013.MUC1 cell lines, the interaction was significantly higher in the MUC1 expressing cells, confirming the Co-IP in A. However, there was no significant difference in interaction in the Panc1.Neo and Panc1.MUC1 cells. (Student’s t-test: *** p < 0.0005).</p
MiR-200c and MUC1 levels in a panel of pancreatic cancer cell lines and tumor samples.
<p>Correlation between levels of MUC1 and miR-200c in a panel of pancreatic cancer cell lines and a panel of primary tumor sections and liver metastases. A) MiR-200c levels, indicated by Ct value, are the white bars and correspond to the left y-axis. MUC1 statuses of these cell lines, determined by western blot, are represented in the black bars and right y-axis. Levels of miR-200c and their corresponding MUC1 levels [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073306#B20" target="_blank">20</a>], on the left and right y-axis respectively were evaluated in primary pancreatic tissue (B) and liver metastases (C). MUC1 status was determined based on IHC staining from one section per patient; therefore standard deviation was not determined.</p
qRT-PCR confirmation of microRNA levels in cells and pancreatic cancer tissue.
<p>MicroRNA levels were evaluated in triplicate by qRT-PCR to confirm expression changes identified by microarray analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073306#pone-0073306-t001" target="_blank">Table 1</a>). MicroRNA levels were normalized to the U6 RNA control. A) MiRs-200c, -141, -192, -33b, -194, -215, and -376c expression levels were evaluated in S2.013.Neo compared to S2.013.MUC1 cells. B) MiRs-200c, -141, -192, -33b, -194, -215, and -376c expression levels were evaluated in Panc1.Neo and Panc1.MUC1 cells. C) Levels of miR-200c were evaluated in a panel of pancreatic cancer tissues. These data are represented by Ct value, where lower Ct values indicate higher levels of miR-200c, and each data point represents a different patient sample. Uninvolved tissue is non-cancerous tissue isolated from a cancer patient. (Student’s t-test: *** p < 0.0005, ** p < 0.005, * p < 0.05).</p
Chromatin Immunoprecipitation confirmation of MUC1.CT and ZEB1 occupancy of the miR-200c/141 promoter.
<p>Chromatin immunoprecipitation (ChIP) was utilized to confirm MUC1.CT and ZEB1 occupancy at the miR-200c/141 promoter region. A) Schematic representing the miR-200c/141 promoter region where the ChIP primer sets were designed (-480 to -210, and +552 to +675) and the known ZEB1 binding region (Grey bar). B) ChIP results indicate fold change of normalized relative levels of MUC1.CT occupancy at the ZEB1 binding region (based on qPCR) of the miR-200c/141 promoter in S2.013.MUC1 cells compared to S2.013.Neo cells. C) ChIP results indicate fold change of normalized relative levels of ZEB1 occupancy at the ZEB1 binding region (based on qPCR) of the miR-200c/141 promoter in S2.013.MUC1 cells compared to S2.013.Neo cells. D) Sequence of the MUC1 cytoplasmic tail, indicating three phosphorylated tyrosine motifs that were examined here. E-F) ChIP results indicate relative levels (based on qPCR and normalized to IgG control) of phospho-YHPM, -YVPP, and -YEKV MUC1 cytoplasmic tail at the ZEB1 binding region of miR-200c/141 in the S2.013.MUC1 cells, E, and Panc1.MUC1 cells, F. In both cell lines, only the phospho-YEKV form of MUC1.CT shows significant enrichment at the promoter. All ChIP data was normalized to antibody specific IgG control. (Student’s t-test: *** p < 0.0005, ** p < 0.005, * p< 0.05).</p
PLA analysis of MUC1.CT interaction with ZEB1 in mitotic cells.
<p>Proximity ligation assay of MUC1.CT with ZEB1 was analyzed in mitotic cells versus non-mitotic cells. A) PLA results from mitotic (circled) or non-mitotic cells in S2.013.Neo or S2.013.MUC1, B indicate a roughly 2-3-fold increase in MUC1.CT and ZEB1 interaction. In addition, this interaction occurs mainly in the cytoplasm, not in the nuclei. (*** p < 0.0005).</p