Figure 2. GLCE expression in human breast tumors and control tissues in vivo. (A and B) GLCE mr and protein levels in the same clinical samples; GLCE expression normalized to that of β-actin and GApDH, respectively. 300–326 patients, control and tumor breast tissues (matched pairs from each patient), bars represent the mean ± SD from triplicate experiments (Originpro 8.1). (C and D) Correlation analysis between mir NA-218 and GLCE mr (C) or GLCE protein (D) expression in breast tissues and tumors in vivo. pearson’s correlation coefficients are shown (Originpro 8.1). cancer.23 In addition, significant loss of miRNA-218 was shown during prostate cancer progression,24 although unchanged miRNA-218 expression was shown in mouse colon tumors.25 In a number of functional studies, a tumor-suppressor role for miRNA-218 was shown in non-small cell lung cancers,13 bladder cancer,14 nasopharyngeal carcinoma15 and renal cell carcinoma.16 However, it is not clear whether miRNA-218 has tumor-sup- pressor activity in human breast tissue/tumors as well. According to our results, miRNA-218 expression significantly decreased in breast tumors, which correlated with GLCE mRNA level both in normal breast tissues (r = +0.45) and breast tumors (r = +0.79). This implicates coordinate expression, which looks logical for GLCE protein regulation—the more GLCE mRNA in the cell, the more miRNA-218 needed to keep GLCE pro- tein at a stable level. Possibly, a feedback loop may exist between miRNA-218 and GLCE expression, as it was recently shown for some other genes.26,27 miRNA-218 negatively regulates expression of GLCE protein in MCF7 breast cancer cells in vitro and primary breast tumors in vivo. However, simultaneous general downregulation of miRNA- 218 and GLCE in breast tumors raises the question of the functional interrelation between a potential tumor-suppressor, miRNA-218, and its downstream target, tumor-suppressor gene GLCE in breast cancer. It is known that the tumor-suppressor activity of miRNA-218 is achieved through the inhibition of cancer-promoting genes related to mTOR-Akt signaling pathway,28 MMP-9 and IKK-β/ NF-κB pathway,20 SLIT-ROBO pathway,15,23,29 transcrip- tion factors HNF-6/OC-1, OC-230 and Runx2,31 some other downstream targets such as paxillin,21 actin filament bundling proteins LIM and SH3 protein 1 (LASP1)32 and epithelial cell- specific marker LAMB3.33 To avoid the tumor-suppressor effect, miRNA-218 seems to be inactivated during the carcinogenesis. However, miRNA-218 inactivation also supposes simultane- ous increase of potential miRNA-218 targeted tumor-suppressor proteins (GLCE, RASSF1A and claudin-634). The upregula- tion of tumor-suppressor genes could counteract malignant transformation and may be suppressed by some other molecu- lar mechanisms, not directly related to miRNA-218. Possibly, a clonal selection of the cells with simultaneous inactivation of both miRNA and its target tumor-suppressor genes is a necessary step for tumor development. The hypothesis is sup- ported by the recent publication on a previously unappreciated miRNA-regulation mechanism by which a single miRNA may target both oncogenes and tumor suppressors, simultaneously or www.landesbioscience.com Epigenetics 1111