Abstract
BackgroundMicroRNA (miRNAs) are non-coding small RNA molecules that regulate gene expression by inhibiting the translation of target mRNAs. Among several dysregulated miRNAs in human cancer, the up-regulation of miR-221 has been associated with development of a variety of hematologic and solid malignancies. In this study, we investigated the involvement of miR-221 in breast cancer.MethodsTaqMan microRNA assay was used to detect the miR-221 levels in normal cells and in MDA-MB 231 and SkBr3 breast cancer cells as well as in main players of the tumor microenvironment, namely cancer-associated fibroblasts (CAFs). miR-221 mimic sequence and locked nucleic acid (LNA)-i-miR-221 construct were used to induce or inhibit, respectively, the miR-221 expression in cells used. Quantitative PCR and western blotting analysis were performed to evaluate the levels of the miR-221 target gene A20 (TNFAIP3), as well as the member of the NF-kB complex namely c-Rel and the connective tissue growth factor (CTGF). Chromatin immunoprecipitation (ChIP) assay was performed to ascertain the recruitment of c-Rel to the CTFG promoter. Finally, the cell growth and migration in the presence of LNA-i-miR-221 or silencing c-Rel and CTGF by specific short hairpin were assessed by cell count, colony formation and boyden chambers assays. Statistical analysis was performed by ANOVA.ResultsWe first demonstrated that LNA-i-miR-221 inhibits both endogenous and ectopic expression of miR-221 in our experimental models. Next, we found that the A20 down-regulation, as well as the up-regulation of c-Rel induced by miR-221 were no longer evident using LNA-i-miR-221. Moreover, we established that the miR-221 dependent recruitment of c-Rel to the NF-kB binding site located within the CTGF promoter region is prevented by using LNA-i-miR-221. Furthermore, we determined that the up-regulation of CTGF mRNA and protein levels by miR-221 is no longer evident using LNA-i-miR221 and silencing c-Rel. Finally, we assessed that cell growth and migration induced by miR-221 in MDA-MB 231 and SkBr3 breast cancer cells as well as in CAFs are abolished by LNAi-miR-221 and silencing c-Rel or CTGF.ConclusionsOverall, these data provide novel insights into the stimulatory action of miR-221 in breast cancer cells and CAFs, suggesting that its inhibition may be considered toward targeted therapeutic approaches in breast cancer patients.
Highlights
MicroRNA are non-coding small RNA molecules that regulate gene expression by inhibiting the translation of target mRNAs
Results miR-221 down-regulates A20 expression in cancer-associated fibroblasts (CAFs), MDA-MB 231 and SkBr3 breast cancer cells On the basis of previous studies showing that miR-221 may act as an oncogenic factor in certain malignancies, we began the present investigation assessing that the levels of the endogenous miR-221 are higher in crucial players of the tumor microenvironment as CAFs and in both MDA-MB 231
MiR-221 prompts the expression of c-Rel and its recruitment to the connective tissue growth factor (CTGF) promoter region in CAFs, MDA-MB 231 and SkBr3 breast cancer cells Previous studies have shown that A20 may regulate nuclear factor kappa B (NF-kB), in particular a negative correlation between the levels of
Summary
MicroRNA (miRNAs) are non-coding small RNA molecules that regulate gene expression by inhibiting the translation of target mRNAs. In bone marrow-derived macrophages, miR-221 was reported to down-regulate the expression of the ubiquitin-editing A20 [16] enzyme, which acts toward the maintenance of tissue homeostasis and the prevention of inflammatory disorders [17]. In this vein, it was demonstrated that A20 inhibits the activity of the nuclear factor kappa B (NF-kB) [18], which is largely involved in the development of many types of tumors [19, 20]. Previous studies have reported its involvement in various malignancies as breast and endometrial tumors [35], melanoma [36], gastric [37], pancreatic [38], prostate [39], hepatic [40] and colon cancer [41]
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