Interestingly, we found that expression

of miR-148a and miR-152, which were silenced in cholangiocarcinoma compared with H69 cells, were further decreased with enforced IL-6 expression both in vitro and in vivo. We further demonstrated that miR-148a and miR-152 can negatively modulate the expression of DNMT-1, by interacting with the 3′-UTR of the gene. In cells transfected BAY 73-4506 chemical structure with these miRNAs, we observed down-regulation of endogenous DNMT-1 protein expression and decreased cell proliferation. Treatment with 5-Aza-CdR, a methylation inhibitor, increased the expression of tumor suppressor genes, thus supporting the regulation by DNMT-1 through promoter methylation mechanisms. Hypermethylation at promoter CpG islands and AZD4547 inactivation of multiple tumor suppressor genes are common in cholangiocarcinoma, and contribute to tumor growth.13 Indeed, the number of methylated CpG island loci in extrahepatic cholangiocarcinoma specimens increases significantly with the stage of the disease and with the presence of node metastasis.23 DNMT-1 has a role in the establishment and regulation of tissue-specific patterns of methylated cytosine residues. DNMT-1 contributes more to maintenance of methylation than to de novo methylase activity, and deregulated expression of DNMT-1 may play a causal role in cellular transformation.24 Aberrant DNMT-1 expression has been detected

ioxilan in several tumors, including liver tumors,25 supporting a role for DNMT-1 in tumorigenesis. Reduced expression of specific miRNAs such as miR-148 and miR-152 can directly modulate the expression of DNMT-1 in cholangiocytes. In human cholangiocarcinoma, loss of expression of these miRNAs by genetic or environmental factors such as IL-6 overexpression can contribute to enhanced

DNMT-1 level with subsequent silencing of expression of critical tumor suppressor genes through altered promoter methylation. Reduced expression of miR-301 in malignant cholangiocytes in vitro suggests a potential role of this miRNA in tumorigenesis, albeit not one that involves modulation of DNMT-1 expression. To gain insight into potential functional effects of miR-301, we performed a gene annotation enrichment analysis of predicted target genes using the Database for Annotation, Visualization and Integrated Discovery.26 KEGG pathway mapping of the results showed enrichment of genes involved in the Wnt, Notch and transforming growth factor β signaling pathways. These observations warrant further study. Rassf1a has been shown to be the most frequently (65%) hypermethylated tumor suppressor gene in cholangiocarcinoma compared with normal cholangiocytes and is silenced both in extrahepatic and intrahepatic bile ducts tumors.13, 23 Rassf1a can mediate the apoptotic effects of Ras and act as a negative Ras effector, inhibiting cell growth and inducing cell death.