Regulation of the T-box transcription factor Tbx3 by the tumour suppressor microRNA-206 in breast cancer

Abstract

Background: The Tbx3 transcription factor is over-expressed in breast cancer, where it has been implicated in proliferation, migration and regulation of the cancer stem cell population. The mechanisms that regulate Tbx3 expression in cancer have not been fully explored. In this study, we demonstrate that Tbx3 is repressed by the tumour suppressor miR-206 in breast cancer cells.

Methods: Bioinformatics prediction programmes and luciferase reporter assays were used to demonstrate that miR-206 negatively regulates Tbx3. We examined the impact of miR-206 on Tbx3 expression in breast cancer cells using miR-206 mimic and inhibitor. Gene/protein expression was examined by quantitative reverse-transcription-PCR and immunoblotting. The effects of miR-206 and Tbx3 on apoptosis, proliferation, invasion and cancer stem cell population was investigated by cell-death detection, colony formation, 3D-Matrigel and tumorsphere assays.

Results: In this study, we examined the regulation of Tbx3 by miR-206. We demonstrate that Tbx3 is directly repressed by miR-206, and that this repression of Tbx3 is necessary for miR-206 to inhibit breast tumour cell proliferation and invasion, and decrease the cancer stem cell population. Moreover, Tbx3 and miR-206 expression are inversely correlated in human breast cancer. Kaplan-Meier analysis indicates that patients exhibiting a combination of high Tbx3 and low miR-206 expression have a lower probability of survival when compared with patients with low Tbx3 and high miR-206 expression. These studies uncover a novel mechanism of Tbx3 regulation and identify a new target of the tumour suppressor miR-206.

Conclusions: The present study identified Tbx3 as a novel target of tumour suppressor miR-206 and characterised the miR-206/Tbx3 signalling pathway, which is involved in proliferation, invasion and maintenance of the cancer stem cell population in breast cancer cells. Our results suggest that restoration of miR-206 in Tbx3-positive breast cancer could be exploited for therapeutic benefit.

Figure 1

Tbx3 mRNA and miR-206 in human breast cancer. (A) Tbx3 mRNA expression in adjacent (Adj.) normal (n=104) and breast tumour (n=1100) samples. Box plot depicting mRNA expression levels for Tbx3 in human breast invasive carcinoma and adjacent normal breast tissue. (B) miR-206 expression in adjacent (Adj.) normal (n=103) and breast tumour (n=1101) samples. Box plot depicting expression levels for miR-206 in human breast invasive carcinoma and adjacent normal breast tissue. Correlation between Tbx3 mRNA and miR-206 expression levels in (C) adjacent normal breast samples (n=75) and (D) breast tumours (n=330), in which each dot corresponds to one patient sample. Spearman score shows significant inverse correlation between Tbx3 and miR-206 levels in both normal breast tissue and breast cancer. (E) The combination of Tbx3 and miR-206 expression is significantly associated with patient survival. Kaplan–Meier graph (n=1182) showing patients exhibiting a combination of high Tbx3 and low miR-206 expression had lower probability of survival when compared with patients exhibiting a combination of low Tbx3 and high miR-206 expression. **P<0.01 between indicated groups was calculated by Welch two-sample t-test.

Figure 2

Tbx3 is a direct target of miR-206 in breast cancer cells, and restoration of miR-206 decreases Tbx3 mRNA and protein expression in breast cancer cells. (A) Putative miR-206 target site within the human Tbx3 3′ UTR as predicted by TargetScan and PicTar algorithms. (B) The miR-206 wild-type binding sequence or its mutated form was inserted into the C terminus of the luciferase gene to generate pMIR-TBX3-3′ UTR or pMIR-TBX3-mut-3′ UTR, respectively. (C) Bar plots showing relative luciferase activity in HEK-293T cells co-transfected with a luciferase reporter construct containing a fragment of the human Tbx3 3′ UTR encompassing the miR-206 binding site (Tbx3 3′ UTR) or empty vector (V), along with miR scrambled control (NC) or miR-206 for 48 h. Relative luciferase units were normalised to control β-gal reporter vector for transfection efficiency. (D) Sequence alignment showing the evolutionary conservation of the 3′ UTR of Tbx3 target site across different species. (E) Bar plots showing relative Tbx3 mRNA expression levels, in ZR75-1, MCF7 and MDA-MB-231 cells transfected with miR scrambled control (NC) or miR-206. Analysis for the Tbx3 message levels was performed by the comparative Ct method. Relative abundance was determined from the Ct values using the 2^−ΔΔCt method after normalisation to GAPDH. Results are representative of at least three independent experiments. (F) Western blot analysis of MDA-MB-231 and MCF7 cells transfected with miR scrambled control (NC), miR-206 or αmiR-206 for 72 h. Beta-actin served as a loading control. Results are representative of three to five independent experiments. Means with error bars representing±s.e.m. **P<0.01; ***P<0.001.

Figure 3

miR-206-mediated downregulation of Tbx3 impacts its targets, inhibits proliferation and promotes apoptosis. (A) Western blot analysis of MCF7 and MDA-MB-231 cells transfected with miR scrambled (NC), miR-206 or αmiR-206 for 72 h. Beta-actin was used as a protein-loading control. (B) Western blot analysis of ZR75-1 and MDA-MB-231 cells transfected with miR scrambled control (NC) or siRNA-Tbx3 (si-Tbx3). Beta-actin was used as a loading control. (C) Bar plots showing per cent colony formation of MDA-MB-231, ZR75-1 and MCF7 cells transfected with miR scrambled control (NC), miR-206, αmiR-206, siRNA-Tbx3 (si-Tbx3) or left untreated (UT). Per cent colony formation was determined with untreated set at 100%. (D) Bar plots showing relative DNA fragmentation in MDA-MB-231, ZR75-1 and MCF7 cells transfected with miR scrambled control (NC), miR-206, αmiR-206, siRNA-Tbx3 (si-Tbx3) or left untreated (UT). Untreated (UT) cells and miR scrambled control (NC) were used as controls. The results are representative of three independent experiment performed in triplicates. Means with error bars representing±s.e.m. *P<0.05; **P<0.01; ***P<0.001.

Figure 4

miR-206 expression and Tbx3 knock-down inhibits the 3D growth of breast cancer cells in Matrigel. Tbx3 lacking the 3′ UTR is not repressed by miR-206. (A) Phase-contrast images of MDA-MB-231 cells in 3D culture treated with miR-206, αmiR-206 or siRNA-Tbx3 (si-Tbx3). Untreated (UT) and miR scrambled control (NC) treated cells were used as controls. (B) Bar plots represent the quantification of the number of invasive colonies, mean colony area and number of branching stellate cells. (C) Western blot analysis of MDA-MB-231 cells co-transfected with miR scrambled control with either empty vector (NC+V) or with flag-tagged TBX3 expression vector (NC+TBX3), and with miR-206 with empty vector (miR-206+V) or with flag-tagged TBX3 expression vector (miR-206+TBX3). Beta-actin was used as a loading control. (D) Phase-contrast images of MDA-MB-231 cells in 3D cultures and co-transfected with miR scrambled control (NC) or miR-206 with either empty vector (V) control or TBX3 expression vector (TBX3). (E) Bar plots showing the reduction in invasive phenotype of MDA-MB-231 cells as quantified by counting invasive colonies, mean colony area and the number of branching stellate cells. The results are representatives of three independent experiments. Scale bar: × 4 images, 200 μm; × 10 images, 50 μm. Means with error bars representing±s.e.m. *P<0.05; **P<0.01; ***P<0.001.

Figure 5

miR-206 exerts a tumour suppressive function and inhibits tumorsphere formation of breast cancer cells. Phase-contrast × 4 images of tumorspheres of (A) MCF7 and (B) MDA-MB-231 cells reverse-transfected with miR scrambled control (NC), miR-206, αmiR-206, siRNA-Tbx3 (si-Tbx3), miR-206 plus empty vector (miR-206+V) or miR-206 plus TBX3 expression vector (miR-206+TBX3). Bar plots showing the relative sphere number of (B) MCF7 and (E) MDA-MB-231 cells corresponding to A and B, respectively. Relative sphere number was normalised to miR scrambled control (NC). Images are representative of spheres under the mentioned treatments at the secondary sphere stage. Western blot analysis of (C) MCF7 and (F) MDA-MB-231 cells co-transfected with miR scrambled control (NC), miR-206, αmiR-206, siRNA-Tbx3 (si-Tbx3), miR-206 plus empty vector (miR-206+V) or miR-206 plus TBX3 expression vector (miR-206+TBX3). Beta-actin was used a loading control. Scale bar: 200 μm. Means with error bars representing±s.e.m. *P<0.05; **P<0.01.