Long-Term Survival of an Elderly Patient with Carcinosarcoma of the Gallbladder after Cholecystectomy

Carcinosarcomas, often referred to as malignant mixed tumors, are rare neoplasm. We reported herein a carcinosarcoma of the gallbladder in an elderly patient with long-term survival (4 years). The operation carried out was open cholecystectomy under the preoperative diagnosis of chronic cholecystitis and tumor of the gallbladder. Anticancer chemotherapy after cholecystectomy was performed by oral low-dose FT therapy. He was alive with no evidence of disease 48 months after surgery. Long-term survival for only cholecystectomy treatment as in this case may be possible if oral low-dose FT anticancer therapy is effective against carcinosarcoma of the gallbladder

<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

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

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