Figure 1. mir NA-218 expression in the human breast tumors and breast tissues in vivo. mir NA-218 expression normalized to that of β-actin, 300–326 patients, control and tumor breast tissues (matched pairs from each patient), bars represent the mean ± SD from triplicate experiments (Originpro 8.1). miRNA-218 downregulation was observed in most of the cor- responding breast tumors (patients NN 300, 301, 302, 308, 317 and 321) resulting in a more homogeneous, low-level expres- sion of miRNA-218 in cancer samples. The results showed that miRNA-218 expression is significantly decreased (2- to 15-fold) in human breast tumors compared with normal breast tissues. Correlation of miRNA-218 and GLCE expression in breast tumors in vivo. To investigate a possible interrelation between miRNA-218 and GLCE expression, GLCE mRNA and protein levels were determined in the same breast tumors and normal tissues by TaqMan-based qReal-time RT-PCR and western blot analysis, respectively (Fig. 2A and B). Pearson correlation coefficients were calculated for miRNA- 218/GLCE mRNA and miRNA-218/GLCE protein levels both in the normal breast tissue and breast primary tumors (Fig. 2C and D). A positive correlation between miRNA-218 and GLCE mRNA levels was shown for both normal breast tissues (r = +0.45, p < 0.23) and breast tumors (r = +0.79, p < 0.01), indi- cating a possible co-regulation for their expressions in breast tis- sue. On the contrary, a negative correlation between miRNA-218 expression and GLCE protein levels was shown for both groups (r = -0.45, p < 0.26 for normal breast tissues and r = -0.40, p < 0.30 for breast tumors). This suggests an involvement of miRNA-218 in post-transcriptional regulation of GLCE protein levels in human breast tissues and tumors in vivo, although a moderate correlation between miRNA-218 and GLCE protein levels in the samples indicates a possible involvement of some other molecular mechanisms as well. miRNA-218 regulates GLCE protein level but not mRNA in breast cancer cells in vitro. To confirm the ability of miRNA-218 to directly regulate GLCE protein content in breast tissue, a func- tional study was performed in MCF7 breast carcinoma cells in vitro. Mimic miRNA-218 and anti-miRNA-218 oligonucleotides were transfected into MCF7 cells and, subsequently, miRNA- 218, GLCE mRNA and GLCE protein levels were analyzed (Fig. 3). The results showed that transfection with either miRNA- 218 or anti-miRNA-218 did not affect GLCE mRNA level in MCF7 cells. However, GLCE protein contents were significantly decreased in the miRNA-218-transfected cells, showing an abil- ity of miRNA-218 to directly regulate GLCE protein level in breast cancer cells in vitro. Interestingly, transfection of MCF7 cells with anti- miRNA-218 resulted in the decreased miRNA-218 content in the cells (by 2–3-fold) but did not increase the GLCE protein level. This supports the existence of some other molecular mechanisms or miRNAs, which cooperate with miRNA-218 in the control of GLCE protein levels. Taken together, these results complement and extend the pub- lished data on miRNA-218 expression in breast cancer, and show an involvement for miRNA-218 in posttranscriptional regulation of GLCE expression in breast cancer cells in vitro and normal breast tissues and primary tumors in vivo. Discussion microRNAs (miRNAs) are small non-coding endogenously pro- duced RNAs that play key roles in controlling the expression of many cellular proteins.17,18 One of the key results of the study is the specific downregu- lation of miRNA-218 in human breast tumors compared with the surrounding normal breast tissues. The results stay in line with the published data on miRNA-218 deregulation in different tumors—a decreased miRNA-218 expression was shown in glio- blastoma,19 glioma cell lines and primary tumors,20 non-small cell lung cancer,21 clear cell renal cell carcinoma22 and gastric 1110 Epigenetics Volume 7 issue 10