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. 2016 May 13:6:25798.
doi: 10.1038/srep25798.

Slug-upregulated miR-221 promotes breast cancer progression through suppressing E-cadherin expression

Yi Pan  1   2 , Jing Li  1   2 , Yaqin Zhang  1   2   3 , Nan Wang  1   2 , Hongwei Liang  1   2 , Yuan Liu  4 , Chen-Yu Zhang  1   2 , Ke Zen  1   2   4 , Hongwei Gu  1   2
Affiliations

Slug-upregulated miR-221 promotes breast cancer progression through suppressing E-cadherin expression

Yi Pan et al. Sci Rep. .

Abstract

It is generally regarded that E-cadherin is downregulated during tumorigenesis via Snail/Slug-mediated E-cadherin transcriptional reduction. However, this transcriptional suppressive mechanism cannot explain the failure of producing E-cadherin protein in metastatic breast cancer cells after overexpressing E-cadherin mRNA. Here we reveal a novel mechanism that E-cadherin is post-transcriptionally regulated by Slug-promoted miR-221, which serves as an additional blocker for E-cadherin expression in metastatic tumor cells. Profiling the predicted E-cadherin-targeting miRNAs in breast cancer tissues and cells showed that miR-221 was abundantly expressed in breast tumor and metastatic MDA-MB-231 cells and its level was significantly higher in breast tumor or MDA-MB-231 cells than in distal non-tumor tissue and low-metastatic MCF-7 cells, respectively. MiR-221, which level inversely correlated with E-cadherin level in breast cancer cells, targeted E-cadherin mRNA open reading frame (ORF) and suppressed E-cadherin protein expression. Depleting or increasing miR-221 level in breast cancer cells induced or decreased E-cadherin protein level, leading to suppressing or promoting tumor cell progression, respectively. Moreover, miR-221 was specifically upregulated by Slug but not Snail. TGF-β treatment enhanced Slug activity and thus increased miR-221 level in MCF-7 cells. In summary, our results provide the first evidence that Slug-upregulated miR-221 promotes breast cancer progression via reducing E-cadherin expression.

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Figures

Figure 1
Figure 1. Restore E-cadherin expression in tumor cells using wild type (WT) E-cadherin-expressing vector, and compared the levels of E-cadherin protein and mRNA transcript at various time points.
(a,b) E-cadherin protein levels (a) and mRNA levels (b) in MCF-7 and MDA-MB-231 cells. (c) Confocal immunofluorescent images of E-cadherin levels in MCF-7 cells and MDA-MB-231 cells. (d,e) E-cadherin mRNA levels (d) and protein levels (e) in MCF-7 and MDA-MB-231 cells transfected with WT E-cadherin-expressing vector (E-cad) for 0, 24, 48 or 72 h. Results are presented as the mean ± SEM (n = 3). *P < 0.05. **P < 0.01.
Figure 2
Figure 2. Posttranscriptional regulation of E-cadherin expression in breast cancer cells by miR-221.
(a) Levels of all predicted miRNAs targeting E-cadherin ORF in paired breast cancer and normal tissue samples. (b) Levels of all predicted miRNAs targeting E-cadherin ORF in MCF-7 and MDA-MB-231 cells. (c) Schematic depiction of the Luciferase reporter consisting of full-length of E-cadherin ORF. The hypothetical duplexes formed by wild-type E-cadherin ORF and miR-221 were indicated. The mutated sites in E-cadherin MUT were marked in red. (d) Luciferase activity in MCF-7 cells transfected with either WT or Mut E-cadherin ORF plus pre-miR-221, anti-miR-221 or scrambled RNA oligonucleotides (control-pre-NTC). Cells were assayed using a luciferase assay kit 24 h post-transfection. (e,f) Overexpression of miR-221 in MCF-7 cells strongly reduced the expression of E-cadherin protein (e) and resulted in small but significant reduction in E-cadherin mRNA levels (f). (g,h) Knockdown of miR-221 level in MDA-MB-231 cells using anti-miR-221 ASO significantly induced E-cadherin protein expression (g) and resulted in E-cadherin mRNA slightly elevation (h). (i) E-cadherin protein levels in MCF-7 cells transfection with or without WT E-cadherin ORF and pre-miR-221. (j) E-cadherin protein levels in MDA-MB-231 cells transfected with or without WT E-cadherin ORF and anti-miR-221 ASO. Results are presented as the mean ± SEM (n = 3). NS, no significant difference, *P < 0.05. **P < 0.01.
Figure 3
Figure 3. MDA-MB-231 cells can express functional E-cadherin that has the same amino acid sequence but not targeted by miR-221.
(a) Construct of the vector encoding a mutated E-cadherin ORF (E-cad MUT) that has the same amino acids with WT E-cadherin but no miR-221 binding site. The mutated sites in E-cadherin MUT were marked in red. (b,c) E-cadherin mRNA levels (b) and protein levels (c) in MCF-7 and MDA-MB-231 cells transfected with E-cad MUT for 0, 24, 48 or 72 h. (d) E-cadherin protein levels in MCF-7 cells transfected with or without E-cad MUT and pre-miR-221. (e) E-cadherin protein levels in MDA-MB-231 cells transfected with or without E-cad MUT and anti-miR-221 ASO. Results are presented as the mean ± SEM (n = 3). NS, no significant difference, *P < 0.05. **P < 0.01.
Figure 4
Figure 4. Upregulation of miR-221 in breast cancer cells by Slug but not Snail.
(a,b) Slug protein levels (a) and SNAI1 protein levels (b) in MCF-7 and MDA-MB-231 cells. (c,d) Relative level of SNAI protein (c) and miR-221 levels (d) in MDA-MB-231 cells after transfection with SNAI1 siRNA or CTL-siRNA. (e,f) Relative level of SNAI protein (e) and miR-221 levels (f) in MDA-MB-231 cells after transfection with Slug siRNA or CTL-siRNA. (g,h) Relative level of Slug protein (g) and miR-221 levels (h) in MCF-7 cells after transfection with Slug-expressing vector (Slug vector) or control vector (CTL vector), (i) TGF-β (2 ng/ml) time-dependently increased the levels of Slug protein in MCF-7 cells. (j) TGF-β (2 ng/ml) time-dependently increased miR-221 level, (k) reduced E-cadherin protein expression in MCF-7 cells. Results are presented as the mean ± SEM (n = 3). *P < 0.05. **P < 0.01.
Figure 5
Figure 5. miR-221 promotes breast cancer cell migration via reducing E-cadherin.
(a)Migration of MCF-7 cells transfected with pre-miR-221 or Pre-miR-CTL. (b) Migration of MDA-MB-231 cells transfected with anti-miR-221 ASO or anti-CTL ASO. (c) Migration of MCF-7 cells transfected with E-cad (WT) ORF, or pre-miR-221. (d) Migration of MDA-MB-231 cells transfected with E-cad (WT) ORF, or pre-miR-221. (e) Migration of MCF-7 cells transfected with E-cad (MUT) ORF, or anti-miR-221 ASO. (f) Migration of MDA-MB-231 cells transfected with E-cad (MUT) ORF, or anti-miR-221 ASO. Results are presented as the mean ± SEM (n = 3). NS, no significant difference, **P < 0.01.
Figure 6
Figure 6. miR-221 promotes breast cancer cell invasion via reducing E-cadherin.
(a) Invasion of MCF-7 cells transfected with pre-miR-221 or scrambled control oligonucleotide (pre-miR-NTC). (b) Invasion of MDA-MB-231 cells transfected with anti-miR-221 ASO or control ASO (anti-CTL ASO). (c) Invasion of MCF-7 cells transfected with or without E-cad (WT) ORF or pre-miR-221. (d) Invasion of MDA-MB-231 cells transfected with or without E-cad (WT) ORF, or anti-miR-221 ASO. (e) Invasion of MCF-7 cells transfected with or without E-cad (MUT) ORF, or pre-miR-221. (f) Invasion of MDA-MB-231 cells transfected with or without E-cad (MUT) ORF or anti-miR-221 ASO. Results are presented as the mean ± SEM (n = 3). NS, no significant difference, *P < 0.05. **P < 0.01.
Figure 7
Figure 7. E-cadherin inhibits the lung colonization of MDA-MB-231 cells.
(a) Experimental design. Immunocompromised mice were injected through tail vein with control MDA-MB-231 cells or MDA-MB-231 cells that were stably transfected with E-cad (WT), E-cad (WT) plus anti-miR-221 or E-cad (Mut). After 8 weeks, mice were sacrificed and lungs were extracted. (b) the numbers of tumor nodules in the lungs. Results were derived from five mice in each group. (c–e) Mouse lungs were subjected to H&E staining (c) and immunohistochemical staining for Ki-67 (d) and E-cadherin (e), respectively. Left panel: representative image; right panel: quantitative analysis of images. Results are presented as the mean ± SEM (n > 3). **P < 0.01.
Figure 8
Figure 8. A working model that illustrates the mechanism by which Slug suppresses E-cadherin at both transcriptional and posttranscriptional level.
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