ZNF281-miR-543 Feedback Loop Regulates Transforming Growth Factor-β-Induced Breast Cancer Metastasis

Abstract

Breast cancer is the most common malignancy, and metastasis is the main cause of cancer-associated mortality in women worldwide. Transforming growth factor-β (TGF-β) signaling, an inducer of epithelial-to-mesenchymal transition (EMT), plays an important role in breast cancer metastasis. Abnormal expression of miR-543 is associated with tumorigenesis and progression of various human cancers; however, the knowledge about the role of miR-543 in breast cancer metastasis is still unknown. In this study, we demonstrated that miR-543 inhibits the EMT-like phenotype and TGF-β-induced breast cancer metastasis both in vitro and in vivo by targeting ZNF281. ZNF281 transactivates the EMT-related transcription factor ZEB1 and Snail. Furthermore, both ZEB1 and Snail can transcriptionally suppress miR-543 expression. Taken together, our data uncover the ZNF281-miR-543 feedback loop and provide a mechanism to extend the understanding of TGF-β network complexity.

Keywords: ZNF281; breast cancer; epithelial-to-mesenchymal transition; metastasis; miR-543; transforming growth factor-β.

Graphical abstract

Figure 1

miR-543 Inhibits Breast Cancer Metastasis (A) The miR-543 expression in breast invasive carcinoma and normal tissues from TCGA database. (B) Kaplan-Meier analysis of the overall survival in patients with different miR-543 expression levels from TCGA database. (C) The expression of miR-543 in breast cancer cell lines and the normal breast cell line by qRT-PCR. (D) The expression of miR-543 in miR-543-overexpressed MDA-MB-231-luc and control cells by qRT-PCR. (E) Transwell analysis of cell invasion in cells as in (D). (F) Bioluminescent imaging and quantification plot of mice harboring lung metastases after tail vein injection of cells as in (D). ∗∗∗p < 0.001, ∗∗p < 0.01.

Figure 2

Depletion of miR-543 Induces an EMT-like Phenotype (A) The expression of miR-543 in MCF10A transfected with miR-543 inhibitors and control cells determined by qRT-PCR. (B) Morphology of cells as in (A). (C) Transwell analysis of cell invasion in cells as in (A). (D) Immunofluorescence analyses of epithelial marker E-cadherin and mesenchymal marker Vimentin in the cells as in (A). (E and F) mRNA (E) and protein (F) expression levels of epithelial markers E-cadherin and ZO-1 and mesenchymal markers N-cadherin and Vimentin in the cells described in (A) were evaluated by qRT-PCR and western blotting, respectively. ∗∗∗p < 0.001.

Figure 3

ZNF281 Is a Target of miR-543 (A) The potential target genes of miR-543 analyzed by starBase v.3.0. (B) The predicted binding of miR-543 with ZNF281 3ʹ UTR. (C) A dual-luciferase reporter assay was performed to validate the interaction between miR-543 and ZNF281. (D and E) The expression levels of ZNF281 in miR-543-overexpressed MDA-MB-231 (left) or miR-543-depleted MCF10A cells (right), as well as the control cells by qRT-PCR (D) and western blotting (E). ∗∗∗p < 0.001.

Figure 4

miR-543 Inhibits Breast Cancer Metastasis by Regulation of ZNF281 (A) The expression of miR-543 in miR-543- or ZNF281/miR-543-overexpressed MDA-MB-231, as well as the control cells determined by qRT-PCR. (B) Morphology of cells as in (A). (C) The protein expression levels of epithelial markers E-cadherin and ZO-1, mesenchymal markers N-cadherin and Vimentin, and ZNF281 in the cells described in (A) were evaluated by western blotting. (D) Transwell analysis of cell invasion in cells as in (A). (E) Immunofluorescence analyses of epithelial marker E-cadherin and mesenchymal marker Vimentin in the cells as in (A). (F) The mRNA expression levels of epithelial markers E-cadherin and ZO-1 and mesenchymal markers N-cadherin and Vimentin in the cells described in (A) were evaluated by qRT-PCR. ∗∗∗p < 0.001.

Figure 5

ZNF281 Transactivates the Snail and ZEB1 Expression (A) The expression of EMT-related factors in ZNF281-overexpressed MCF10A and control cells. (B) Schematic depiction of the ZEB1 and SNAIL promoter. Black bars indicated primers for ChIP analysis. (C and D) ChIP analysis of interaction between ZNF281 and the ZEB1 (C) or Snail (D) promoter in ZNF281-transfected MCF10A and control cells using anti-HA or anti-IgG antibodies. Results represent the percentage of input chromatin. (E and F) Dual-luciferase analysis of ZEB1 (E) or Snail (F) promoter activity in ZNF281-transfected 293FT and control cells. ∗∗∗p < 0.001, ∗p < 0.01.

Figure 6

The Feedback Loop of ZNF281-Snail/ZEB1-miR-543 (A) Scheme of the miR-543 promoter and ZEB1/Snail binding sites. Black bars indicated primers for ChIP analysis. (B) ChIP analysis of interaction between ZEB1/Snail and the miR-543 promoter in MDA-MB-231 cells using anti-ZEB1, anti-Snail, or anti-IgG antibodies. Results represent the percentage of input chromatin. (C) Dual-luciferase analysis of indicated miR-543 promoter activity in ZEB1 or Snail-transfected 293FT and control cells. (D) The expression of Snail and miR-543 in Snail-depleted MDA-MB-231 (left), Snail-overexpressed MCF10A (right), as well as the control cells determined by western blotting and qRT-PCR, respectively. (E) The expression of ZEB1 and miR-543 in ZEB1-depleted MDA-MB-231 (left), ZEB1-overexpressed MCF10A (right), as well as the control cells determined by western blotting and qRT-PCR, respectively. (F) The expression of ZNF281, Snail, ZEB1, and miR-543 in ZNF281-depleted MDA-MB-231 (left), ZNF281-overexpressed MCF10A (right), as well as the control cells determined by western blotting and qRT-PCR, respectively. ∗∗∗p < 0.001. P-M1, E-box-1 mutated; P-M2, E-box-2 mutated; P-M1+2, both E-box-1 and -2 mutated; P-wt, wild-type.

Figure 7

ZNF281-miR-543 Feedback Loop Is Involved in TGF-β-Induced EMT (A) The expression of miR-543 in MCF10A cells after treatment with TGF-β1 at indicated concentration. (B) Transwell analysis of miR-543-overexpressed MCF10A or control cells with TGF-β1 and/or ZNF281 overexpression. (C) Luciferase reporter analysis of TGF-β signaling activity in cells as in (B). (D) Localization of SMAD2 in cells as in (B) as determined by immunofluorescence staining. (E) The expression of pSMAD2 in cells as in (B) by western blotting. (F) A model for the ZNF281-miR-543 feedback loop in TGF-β-induced breast cancer metastasis.