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miR-221 stimulates breast cancer cells and cancer-associated fibroblasts (CAFs) through selective interference with the A20/c-Rel/CTGF signaling

Methods: TaqMan 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 andmigration 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.

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