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

Abstract

Background: MicroRNA (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.

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 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.

Results: We 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.

Conclusions: Overall, 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.

Keywords: A20; Breast cancer; C-Rel; CAFs; CTGF; miR-221.

Conflict of interest statement

Ethics approval and consent to participate

Signed informed consent from all the patients was obtained and all samples were collected, identified and used in accordance with approval by the Institutional Ethical Committee Board (Regional Hospital, Cosenza, Italy).

Competing interests

The authors declare that they have no competing interest.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

miR-221 regulates A20 expression in CAFs and breast cancer cells. a Expression of miR-221 in fibroblasts, CAFs, MCF10A, MDA-MB 231 and SkBr3 cells. Raw Ct data were normalized to the housekeeping RNU6 levels and expressed as ΔCt values using the comparative cross threshold (Ct) method. b miR-221 expression in CAFs, MDA-MB 231 and SkBr3 breast cancer cells after transfection for 48 h with 25 nM miR-Ctrl and 25 nM miR-221, alone or in combination with 100 nM LNA-i-miR-221 (LNA-i). c Schematic alignment between the miR-221 sequence and the 3’-UTR mRNA region of A20. d A20 mRNA expression in CAFs, MDA-MB 231 and SkBr3 cells transfected for 48 h with 25 nM miR-Ctrl and 25 nM miR-221, alone or in combination with 100 nM LNA-i-miR-221 (LNA-i). A20 protein expression in CAFs (e), MDA-MB 231 (f) and SkBr3 (g) cells transfected for 48 h with 25 nM miR-Ctrl and 25 nM miR-221, alone or in in combination with 100 nM LNA-i-miR-221 (LNA-i); β-actin serves as a loading control. Below panels show densitometric analysis of the blots normalized to the loading controls. Each column represents the mean ± SD of three independent experiments performed in triplicate. The data are shown as fold induction respect to cells transfected with miR-Ctrl. (*) indicates p < 0.05 and (**) p < 0.01

Fig. 2

miR-221 prompts the protein expression of c-Rel and its recruitment to the CTGF promoter region. c-Rel protein expression in CAFs (a), MDA-MB 231 (b) and SkBr3 (c) cells transfected for 48 h with 25 nM miR-Ctrl and 25 nM miR-221, alone or in combination with 100 nM LNA-i-miR-221 (LNA-i). β-actin serves as a loading control. Below panels show densitometric analysis of the blots normalized to the loading controls. d Putative NF-kB binding site (capital letters in the rectangle) located within the CTGF promoter sequence (− 625 bp to + 62 bp). The transcriptional start site is indicated as + 1. The position of primers used for ChIP-qPCR analyses is underlined. Recruitment of c-Rel to the NF-kB binding site within the CTGF promoter sequence in CAFs (e), MDA-MB 231 (f) and SkBr3 (g) cells transfected for 48 h with 25 nM miR-Ctrl and 25 nM miR-221, alone or in combination with 100 nM LNA-i-miR-221 (LNA-i). Data were normalized to the Input and reported as fold changes respect to IgG. h Luciferase activity of the CTGF reporter gene in CAFs, MDA-MB 231 and SkBr3 cells transfected for 48 h with 25 nM miR-Ctrl and 25 nM miR-221, alone or in combination with 100 nM LNA-i-miR-221 (LNA-i). i Luciferase activity of the CTGF reporter gene in CAFs, MDA-MB 231 and SkBr3 cells transfected for 8 h with shRNA or shRel and then transfected for 48 h with 25 nM miR-221. The luciferase activity was normalized to the internal transfection control; values of cells receiving scrambled controls were set as 1-fold induction upon which the activity obtained with the indicated effectors was calculated. Each column represents the mean ± SD of three independent experiments performed in triplicate. (**) indicates p < 0.01

Fig. 3

c-Rel is involved in the up-regulation of the CTGF triggered by miR-221. a CTGF mRNA levels in CAFs, MDA-MB 231 and SkBr3 cells transfected for 48 h with 25 nM miR-Ctrl and 25 nM miR-221, alone or in combination with 100 nM LNA-i-miR-221 (LNA-i). Data are shown as fold changes respect to the scrambled controls. b CTGF mRNA levels in CAFs, MDA-MB 231 and SkBr3 cells transfected for 8 h with shRNA or shRel and then transfected for 48 h with 25 nM miR-221. Data are shown as fold changes respect to the scrambled controls. CTGF protein levels in CAFs (c), MDA-MB 231 (d) and SkBr3 (e) cells transfected for 48 h with 25 nM miR-Ctrl and 25 nM miR-221, alone or in combination with 100 nM LNA-i-miR-221 (LNA-i). β-actin serves as a loading control. Below panels show densitometric analysis of the blots normalized to the loading controls. CTGF protein levels in CAFs (f), MDA-MB 231 (g) and SkBr3 (h) cells transfected for 8 h with shRNA or shRel and then transfected for 48 h with 25 nM miR-221. β-actin serves as a loading control. Below panels show densitometric analysis of the blots normalized to the loading controls. Efficacy of c-Rel silencing in CAFs (i), MDA-MB 231 (j) and SkBr3 (k) cells. β-actin serves as a loading control. Side panels show densitometric analysis of the blots normalized to the loading controls. Results shown are representative of at least three independent experiments. (**) indicates p < 0.01

Fig. 4

miR-221 induces proliferative effects in CAFs, MDA-MB 231 and SkBr3 cells. Cell proliferation in (a) CAFs, (d) MDA-MB 231 and (g) SkBr3 cells transfected every 2 days with 25 nM miR-Ctrl and 25 nM miR-221, alone or in combination with 100 nM LNA-i-miR-221 (LNA-i) and then counted on day 6. Cell proliferation in CAFs (b) MDA-MB 231 (e) and SkBr3 (h) cells transfected with shRNA or shRel for 8 h and then transfected for 48 h with 25 nM miR-221. Cell proliferation in CAFs (c), MDA-MB 231 (f) and SkBr3 (i) cells transfected with shRNA or shCTGF for 8 h and then transfected for 48 h with 25 nM miR-221. The transfections were renewed every 2 days and cells were counted on day 6. Each data point is the mean ± SD of three independent experiments performed in triplicate. (**) indicates p < 0.01

Fig. 5

miR-221 promotes colony formation in CAFs, MDA-MB 231 and SkBr3 cells. Colony formation in CAFs (a, b), MDA-MB 231 (c, d) and SkBr3 (e, f) cells transfected every 2 days as indicated, after 10 days of incubation cell colonies were stained and pictures were captured by a digital camera. Colonies were counted using the program WCIF ImageJ for Windows. Each data point is the mean ± SD of three independent experiments performed in triplicate. (**) indicates p < 0.01

Fig. 6

miR-221 triggers migratory effects in CAFs, MDA-MB 231 and SkBr3 cells. Cell migration as evaluated by Boyden Chamber assay in (a) CAFs, (c) MDA-MB 231 and (e) SkBr3 cells transfected for 48 h with 25 nM miR-Ctrl and 25 nM miR-221, alone or in combination with 100 nM LNA-i-miR-221 (LNA-i). Cell migration, evaluated by Boyden Chamber assay, in CAFs (b) MDA-MB 231 (d) and SkBr3 (f) cells transfected with shRNA or shRel for 8 h and then transfected for 48 h with 25 nM miR-221. Cells were counted in at least 10 random fields at 10× magnification (Scale bar =200 μm) in three independent experiments performed in triplicate. (**) indicates p < 0.01