Background: The inflammatory cytokine Interleukin-6 (IL-6) is over-expressed in human cholangiocarcinoma and contributes to tumor cell growth. We have recently shown that over-expression of IL-6 can modulate expression of methylation-dependent genes and moreover, can alter the expression of specific microRNAs (miRNAs) involved in tumor growth. Thus, we postulated that IL-6 could regulate miRNA expression via effects on DNA methylation.

Methods: MzChA-1 malignant cholangiocytes were stably transfected to over-express IL-6. Stable transfectants or their respective controls were treated with 5-aza-CdR, an inhibitor of DNA methylation, and miRNA profiling was performed by microarray analysis. The expression of specific miRNAs was confirmed by real-time PCR analysis. Western blot analysis for putative miRNA targets was performed on tumor cells in vitro, or in tumor cell xenografts in vivo.

Results: The expression of the methyltransferases Dnmt-1 and HASJ4442 was increased in IL-6 over-expressing cells compared to MzChA-1 controls (p= 0.002 and 0.0002, respectively). Incubation with 5-aza-CdR decreased the expression of both Dnmt-1 (p=0.003) and HASJ4442 (p<0.001). The expression of 42 miRNAs was up-regulated by 5-aza-CdR treatment and silenced by IL-6. Ofthese 7 miRNAs were > 2-fold up-regulated by 5-aza-CdR treatment plus further significantly down-regulated < 0.4-fold by IL-6 over-expression (p<0.01). One of these, miR-370, is embedded in a CpG island and is highly induced from its own promoter after treatment with chromatin-modifying drug. miR-370 was up-regulated ~ 2.1 fold after treatment with 5-aza-CdR in malignant cholangiocytes, but unchanged in non-malignant cholangiocytes. In IL-6 over-expressing tumor cells, miR-370 expression was decreased by 85 ± 7%. Furthermore, incubation with 5-aza-CdR decreased the expression of the oncogene MAP3K8, a putative target of miR-370 in Mz-ChA-1 cells. Over-expression of IL-6 re-instated MAP3K8 expression and reduced miR-370 expression both in vitro as well as in tumor cell xenografts in vivo.

Summary and conclusions: We have identified selected miRNAs such as miR-370 that may be regulated by a methylation-dependent pathway. Enhanced IL-6 expression may contribute to tumor growth in cholangiocarcinoma by altering methylation and down-regulating the expression of tumor-suppressing miRNA.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA