sequentially, in tumorigenesis and normal development.35 From the presented results and our pre- vious study,11 a possible molecular mech- anism of GLCE inactivation in breast tumors, and its relation to miRNA-218, could be suggested (Fig. 4). In normal breast tissue, GLCE expression is transcrip- tionally regulated by the direct interaction of TFC4/b-catenin transcription complex with the GLCE promoter; GLCE protein level is then adjusted by miRNA-218 at the posttranscriptional level. In breast tumors, TCG4/b-catenin-GLCE promoter interac- tion is blocked due to overall chromatin inactivation at the GLCE promoter site, resulting in the decreased GLCE mRNA and protein levels, in spite of the impair- ment of the regulating effect of miRNA- 218.11 The scheme suggests a multilevel mechanism for GLCE regulation in normal breast tissue and a model for its deregula- tion in breast tumors. Materials and Methods Patients and tissue samples. All tissue samples were obtained from primary breast tumors during the radical surgery at Central Municipal Hospital N1, Novosibirsk, Russia, “snap-frozen” in liquid nitrogen and stored at -70°C. Regions were manually dis- sected from the frozen blocks to provide a consistent tumor cell content of more than 70% in tissues used for analysis. The preva- lent histological type of tumors was duct infiltrating adenocarci- noma of different degree of malignancy. Most patients were at the second stage of malignancy progression according to the TNM formula. All patients provided written informed consent and the study protocol was approved by the Local Ethics Committee in accordance with the Helsinki Declaration of 1975. Cell lines, cell culture and miRNA-218 or anti-miRNA-218 transfection. The human breast cancer cell line MCF7 was obtained from the Karolinska Institute. Cells were maintained in IMDM medium supplemented with 2mM L-glutamine, 100 units penicillin, 100 μg/ml streptomycin and 10% (v/v) fetal bovine serum at 37°C in a humidified 5% CO2. For analysis, cells were harvested using trypsin/EDTA (Sigma). MCF7 cells were transfected with miRNA-218 and anti- miRNA-218 oligonucleotides as described previously.9 Briefly, miRNA-218 LNA-enhanced oligonucleotides possessing 2-O- Me modifications on the outermost 6 nucleotides (Exiqon, final concentration of 10 nmol/L) or anti-miRNA-218 LNA-modified oligonucleotides (final concentration of 10 nmol/L) were trans- fected using Lipofectamine 2000 (Invitrogen). Forty-eight hours later, total RNA or protein was isolated. Sequences were as fol- lows: miR-218, 5-AAC CAC ATG GTT AGA TCA AGC ACA A-3; anti–miR-218, 5-GTT AGA TCA AGC ACA-3. Analyses of miRNA-218 expression using the TaqMan MicroRNA assay. Total RNA was extracted from the cells using 1112 Epigenetics Volume 7 issue 10 Figure 3. t ransfection of MCf7 breast carcinoma cells with mimic mi r NA-218 and anti-mir NA-218 oligonucleotides. mir NA-218 expression, t aqMan Micror Assay; GLCE mr expression, t aqman-based qr eal-t ime rt -pCr ; GLCE protein level, western blot. inset: a representative blot. Bars represent the mean ± SD from triplicate experiments (Originpro 8.1). the TRIZOL reagent (Invitrogen) according to the manufac- turer’s instructions. miRNA-218 quantification was performed using the BioRad IQ5 Multicolor Real-Time PCR Detection System (BioRad) and the two-step RT-PCR TaqMan MicroRNA assay (Invitrogen). At the reverse transcription (RT) step, cDNA was synthesized from total RNA samples using specific miRNA primers and reagents from the TaqMan MicroRNA Reverse Transcription Kit. At the PCR step, PCR products were ampli- fied from cDNA using the TaqMan MicroRNA Assay/TaqMan Universal PCR Master Mix. The following conditions were used for PCR: 95°C, 10 min; 95°C, 15 sec; 60°C, 60 sec; 40 cycles. β-actin (ACTB) was used as the housekeeping gene. Analyses of GLCE expression by quantitative TaqMan- based real-time RT-PCR. Quantitative real-time RT-PCR (qRT- PCR) analysis of GLCE expression was performed as described earlier.11,12 Briefly, total RNA was extracted from the cells using the TRIZOL reagent (Invitrogen), cDNA was synthesized from 1–2 μg of total RNA using a First Strand cDNA Synthesis kit (Fermentas), and 1/10th of the product was subjected to PCR analysis. qRT-PCR was performed using the BioRad IQ5 Multicolor Real-Time PCR Detection System (BioRad) and the GLCE TaqMan Custom Assay (Applied Biosystems) under the following conditions: 95°C for 3 min, followed by 40 cycles at 95°C for 10 sec and 60°C for 30 sec. The total reaction volume was 25 μl. β-actin (ACTB) was used as the control housekeep- ing gene. The PCR primers and TaqMan probes used were: GLCE-F, 5'-TTC CAA AGT CTA TGC ACA GAG AGC- 3'; GLCE-R, 5'-TCC ACA TTG TAG CCT TCA AAA GAC A-3'; GLCE-probe, 5'-FAM-CCC CTA TCA CCC CGA TGG T-TAMRA-3'; β-actin-F, 5'-GGC ACC CAG CAC AAT GAA G-3', β-actin-R, 5'-GCC GAT CCA CAC GGA GTA CT-3', β-actin-probe, 5'-FAM-TCA AGA TCA TTG CTC CTC CTG AGC GC-TAMRA-3'. Western blotting. Western blotting was performed as described earlier.11 Briefly, cells were lysed with RIPA-buffer (1% Nonidet P-40, 150 mM NaCl, 0.1% SDS, 50 mM Tris, pH 7.4) containing “complete” Protease Inhibitor Cocktail (Roche),