BriEf rEpOrt Epigenetics 7:10, 1109–1114; October 2012; © 2012 Landes Bioscience BriEf rEpOrt miRNA-218 contributes to the regulation of D-glucuronyl C5-epimerase expression in normal and tumor breast tissues t atiana Y. prudnikova,1 Luydmila A. Mostovich,1 Vladimir i. Kashuba,2,3 ingemar Ernberg,3 Eugene r . Zabarovsky3,4 and Elvira V. Grigorieva1,3,* 1institute of Molecular Biology and Biophysics; Siberian Department of r ussian Academy of Medical Sciences; Novosibirsk, r ussia; 2institute of Molecular Biology and Genetics; Ukrainian National Academy of Sciences; Kiev, Ukraine; dDepartment of Microbiology; tumor and Cell Biology (Mt C); Karolinska institute; Stockholm, Sweden; 4Linkoping University; institute of Clinical and Experimental Medicine; Linkoping, Sweden Keywords: miRNA, D-glucuronyl C5-epimerase, GLCE, tumor-suppressor gene, expression, heparan sulphate proteoglycan, biosynthesis, breast cancer micror NAs (mir NAs) are key posttranscriptional regulators of gene expression. in the present study, regulation of tumor-suppressor gene D-glucuronyl C5-epimerase (GLCE) by mir NA-218 was investigated. Significant downregulation of mir NA-218 expression was shown in primary breast tumors. Exogenous mir NA-218/anti-mir NA-218 did not affect GLCE mr but regulated GLCE protein level in MCf 7 breast carcinoma cells in vitro. Comparative analysis showed a positive correlation between mir NA-218 and GLCE mr NA, and negative correlation between mir NA-218 and GLCE protein levels in breast tissues and primary tumors in vivo, supporting a direct involvement of mir NA-218 in posttranscriptional regulation of GLCE in human breast tissue. A common scheme for the regulation of GLCE expression in normal and tumor breast tissues is suggested. Introduction D-glucuronyl C5-epimerase (GLCE, NM_015554) is one of the key enzymes in biosynthesis of heparan sulfate proteogly- cans (HSPGs), which are involved in cell-cell and cell-matrix interactions, intercellular signaling and appropriate cell micro- environment.1-4 Recently, it was shown that GLCE possesses a tumor-suppressor activity in breast and small-cell lung cancer4,5 and GLCE expression is significantly downregulated in primary breast tumors and in breast and lung cancer cell lines compared with controls.6,7 However, the molecular mechanisms of GLCE inactivation in cancer cells remain unclear. Previous studies show that the b-catenin-TCF4 transactivation complex regulates GLCE expression in human colon cancer cells in vitro,8 miR-218 directly represses expression of GLCE in DLD human colon cancer cells in vitro and rat retina in vivo,9 and that overexpression of the EJ-ras oncogene decreases D-glucuronosyl C-5 epimerase mRNA levels in rabbit endothelial cells.10 All the results provide important background for complex study of the molecular mechanisms of GLCE inactivation in breast cancer. We recently showed complex transcriptional regulation for GLCE expression in breast cancer, where the combined effects of chromatin structure and TCF4/β-catenin complex (but not promoter methylation) are necessary to maintain GLCE expres- sion.11 However, a possible contribution of some other molecular *Correspondence to: Elvira Grigorieva; Email: elv_grig@yahoo.com Submitted: 07/22/12; Accepted: 09/07/12 http://dx.doi.org/10.4161/epi.22103 mechanisms was indicated as well. Based on previous observa- tions,9 miRNA-218, as post-transcriptional regulator of GLCE, is a strong candidate to be studied. So far, no functional interrelation for miRNA-218 and GLCE in breast cancer has been demonstrated. On the one hand, tumor-suppressor function was shown both for GLCE5,12 and miRNA-218.13-16 On the other hand, miRNA-218 is supposed to negatively regulate GLCE expression in cancer cells.9 In the present study, miRNA-218 expression levels were deter- mined in primary breast tumors and control breast tissues, and the contribution of miRNA-218 in posttranscriptional regulation of GLCE expression in breast cancer cells in vitro and primary tumors in vivo was investigated. Results miRNA-218 expression is downregulated in human primary breast tumors in vivo. miRNA-218 expression in primary breast tumors and normal breast tissues (morphologically normal, max- imally distant part of the same mammary gland) was determined by the TaqMan MicroRNA Assay (Invitrogen), which uses looped-primer RT-PCR real-time quantification to accurately detect mature miRNAs (Fig. 1). According to the RT-PCR data, miRNA-218 expression was very heterogeneous in normal breast tissues. However, evident www.landesbioscience.com Epigenetics 1109