Real-Time PCR-Based Analysis of the Human Bile MicroRNAome Identifies miR-9 as a Potential Diagnostic Biomarker for Biliary Tract Cancer

Biliary tract cancer (BTC) is often difficult to diagnose definitively, even through histological examination. MicroRNAs (miRNAs) regulate a variety of physiological processes. In recent years, it has been suggested that profiles for circulating miRNAs, as well as those for tissue miRNAs, have the potential to be used as diagnostic biomarkers for cancer. The aim of this study was to confirm the existence of miRNAs in human bile and to assess their potential as clinical biomarkers for BTC. We sampled bile from patients who underwent biliary drainage for biliary diseases such as BTC and choledocholithiasis. PCR-based miRNA detection and miRNA cloning were performed to identify bile miRNAs. Using high-throughput real-time PCR-based miRNA microarrays, the expression profiles of 667 miRNAs were compared in patients with malignant disease (n = 9) and age-matched patients with the benign disease choledocholithiasis (n = 9). We subsequently characterized bile miRNAs in terms of stability and localization. Through cloning and using PCR methods, we confirmed that miRNAs exist in bile. Differential analysis of bile miRNAs demonstrated that 10 of the 667 miRNAs were significantly more highly expressed in the malignant group than in the benign group at P<0.0005. Setting the specificity threshold to 100% showed that some miRNAs (miR-9, miR-302c*, miR-199a-3p and miR-222*) had a sensitivity level of 88.9%, and receiver-operating characteristic analysis demonstrated that miR-9 and miR-145* could be useful diagnostic markers for BTC. Moreover, we verified the long-term stability of miRNAs in bile, a characteristic that makes them suitable for diagnostic use in clinical settings. We also confirmed that bile miRNAs are localized to the malignant/benign biliary epithelia. These findings suggest that bile miRNAs could be informative biomarkers for hepatobiliary disease and that some miRNAs, particularly miR-9, may be helpful in the diagnosis and clinical management of BTC

Sequencing and Bioinformatics-Based Analyses of the microRNA Transcriptome in Hepatitis B–Related Hepatocellular Carcinoma

MicroRNAs (miRNAs) participate in crucial biological processes, and it is now evident that miRNA alterations are involved in the progression of human cancers. Recent studies on miRNA profiling performed with cloning suggest that sequencing is useful for the detection of novel miRNAs, modifications, and precise compositions and that miRNA expression levels calculated by clone count are reproducible. Here we focus on sequencing of miRNA to obtain a comprehensive profile and characterization of these transcriptomes as they relate to human liver. Sequencing using 454 sequencing and conventional cloning from 22 pair of HCC and adjacent normal liver (ANL) and 3 HCC cell lines identified reliable reads of more than 314000 miRNAs from HCC and more than 268000 from ANL for registered human miRNAs. Computational bioinformatics identified 7 novel miRNAs with high conservation, 15 novel opposite miRNAs, and 3 novel antisense miRNAs. Moreover sequencing can detect miRNA modifications including adenosine-to-inosine editing in miR-376 families. Expression profiling using clone count analysis was used to identify miRNAs that are expressed aberrantly in liver cancer including miR-122, miR-21, and miR-34a. Furthermore, sequencing-based miRNA clustering, but not individual miRNA, detects high risk patients who have high potentials for early tumor recurrence after liver surgery (P = 0.006), and which is the only significant variable among pathological and clinical and variables (P = 0,022). We believe that the combination of sequencing and bioinformatics will accelerate the discovery of novel miRNAs and biomarkers involved in human liver cancer

<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

<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

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

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