We have recently shown that IL-6 contributes to tumor growth by m

We have recently shown that IL-6 contributes to tumor growth by modulation of expression of selected

microRNAs (miRNAs).6 miRNAs are important mediators of posttranscriptional regulation of messenger RNA (mRNA) expression and have been shown to modulate the expression of DNMT-3a and DNMT-3b, de novo methyltransferases involved in methylation of DNA during early development.10, 11 In contrast, the modulation of DNMT-1, which is involved in maintenance methylation, is unknown. Several tumor suppressor genes such as Rassf1a and p16INK4 have been shown to be modulated by promoter ABT263 hypermethylation in cholangiocarcinoma.12–15 Thus, we sought to evaluate the potential role of IL-6–mediated changes in miRNA expression as a mechanism of modulation of DNMT-1 expression, and subsequently methylation-dependent regulation of oncogene or tumor suppressor gene expression in cholangiocarcinoma. 5-Aza-CdR, 5-Aza-2′-deoxycytidine; DNMT-1, DNA methyltransferase-1; IL-6, interleukin-6; miRNA, microRNA; mRNA, messenger RNA; UTR, untranslated region. KMCH-1, Mz-ChA-1, and TFK-1 human cholangiocarcinoma cell lines and the nonmalignant human cholangiocyte H69 cell line were obtained

as described.16 Mz-ChA-1 cells are derived from metastatic gallbladder cancer, TFK-1 cells from Epigenetics inhibitor common bile duct cancer, and KMCH-1 from an intrahepatic mixed cholangiocellular–hepatocellular carcinoma. H69 cells are derived from nonmalignant cholangiocytes and immortalized by SV40 transfection. Mz-ChA-1 and TFK-1 cells were cultured in CMRL 1066 medium with 10% fetal bovine serum, 1% L-glutamine, and 1% Edoxaban antimycotic antibiotic

mix. H69 and KMCH-1 cells were cultured in Dulbecco’s modified Eagle medium/F-12 as described.16 All other cell culture media and supplements were obtained from Invitrogen (Carlsbad, CA). For methylation-specific activation or inhibition studies, Mz-ChA-1 and KMCH-1 malignant cholangiocytes were stably transfected with full-length IL-6 to generate cell lines that overexpressed IL-6 (Mz-IL-6 and KM-IL-6) as described.3 To assess 5-Aza-2′-deoxycytidine (5-Aza-CdR) methylation inhibitory effects, cells were grown to 75% confluency on 100-mm culture dishes and then treated with 5 μM 5-Aza-CdR or diluent (acetic acid) control for 24 hours at 37°C. Following treatment, cells were washed twice with cold phosphate-buffered saline before harvesting for isolation of total RNA or protein. Transfections were performed by electroporation using the Nucleofector system (Amaxa Biosystems, Koln, Germany). All studies were performed in quadruplicate. Cells (1 to 2 × 106) were spun down at 1,000 rpm for 5 minutes, and the medium was removed. Cells were then resuspended in 100 μL Nucleofector solution (Amaxa Biosystems) at room temperature followed by addition of 100 nmol/L miRNA precursor or controls (all obtained from Ambion Inc., Austin, TX).

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