The authors declare that they have no competing interests.
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FIGURE LEGENDS
Figure 1: Decreased miR-30a levels in metastatic breast cancer. (A) Comparison of miR-30a levels among normal breast epithelial cells (H184B5F5/M10 and MCF-10A) and breast cancer cell lines that are non-metastatic (BT-474 and MCF-7) or metastatic (Hs578T and MDA-MB-231). miR-30a was quantified by TaqMan real-time PCR, and the relative levels of miR-30a were normalized to RNU6B. (B) Lentiviral transduction with plemiR-30a and subsequent miR-30a overexpression in breast cancer cell lines.
miR-30a levels are expressed as the mean ± SD from three independent experiments.
(C) Western blot showing protein expression of plemiR-30a in E-cadherin–deficient breast cancer cell lines in (B), with -actin as the loading control. (D) Representative images of the phenotypic change from mesenchymal to cobblestone-like epithelial cells in MDA-MB-231 and Hs578T cells transfected with plemiR-30a or plemiR (negative control). Scale bar = 50 μm. (E) Representative phenotypic change in MCF-7 cells transfected with an inhibitor against miR-30a (anti-miR-30a) and negative control (NC) (F) Representative scratch/wound healing assay images for MCF-7 cells were taken at 0 and 24 hr after scarification. (G) Quantification of wound healing area for MCF-7 cells as in (F). Data are expressed as the mean ± SD from triplicate experiments. ***P <
0.001 compared with the control group.
Figure 2: Identification of Slug as a downstream target for miR-30a. (A) Predicted 1
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binding sites for miR-30a within the 3’-UTR of Slug mRNA. The 3’-UTR of Slug contains two binding regions for miR-30a (in red) across different vertebrate species.
(B) Schematic representation of the luciferase reporter constructs showing the sequences at sites 1 and 2 of the three mutants (shown in red) with a mismatch of the miR-30a complementary sequence at site 1 (Slug UTR/mut1), site 2 (Slug 3’-UTR/mut2), or both sites (Slug 3’-UTR/mut3). The wild-type miR-30a-binding sequences are underlined. (C) Luciferase activity was evaluated in Hs578T cells expressing the constructs shown in (B). Firefly luciferase activity was normalized to Renilla luciferase activity and compared with the expression in cells transfected with the pcDNA3 empty vector (control). Data are presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01. (D) Western blot showing Slug expression in breast cancer cell lines transfected with plemiR-30a. β-actin was used as the loading control.
Figure 3: Claudin expression is enhanced by the miR-30a/Slug axis. (A) Schematic of the E-boxes in the promoter regions of human CLDN1, CLDN2, and CLDN3 genes.
The starting point (+1) indicates the transcription initiation in the open reading frame (ORF) of the gene. (B) Western blotting (anti-Slug) of MDA-MB-231 breast cancer cell lysates after lentiviral transduction of miR-30a (left panel). PCR analysis of the genes 1
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encoding CLDN-1, -2, and -3 after ChIP in the presence of anti-Slug or anti-IgG from control MDA-MB-231/plemiR cells (con) and MDA-MB-231/plemiR-30a cells (right panel). (C) Western blotting revealed that Slug and fascin expression in miR-30a–
overexpressing cells following miR-30a knockdown (anti-miR-30a) was inversely correlated with claudin expression. NC, negative control. (D) Microfilaments in MDA-MB-231 and Hs578T cells expressing plemiR-30a or the control construct plemiR were detected with Alexa Fluor 488–conjugated phalloidin (green) as indicated by red
arrows. (E) The number of filopodial tips per cell as averaged from 50 cells per condition was calculated. The data represent the mean ± SD from three independent experiments. ***P < 0.001. (F) Expression of CLDN-1, -2, or -3 (green) was distributed around the cell boundary (white arrowheads) in breast cancer cell clones stably
expressing miR-30a, but not in those expressing plemiR. Nuclei were counterstained in (D) and (F) with DAPI (blue). Scale bar = 20 μm.
Figure 4: miR-30a decreases the invasiveness of breast cancer cells. (A) MDA-MB-231 cells were transfected with plemiR or plemiR-30 and then treated with miR-30a inhibitor (anti-miR-30a) or underwent Slug knockdown with a Slug-specific short hairpin RNA (sh-Slug) or sh-Luc, a control shRNA. The cells were plated in modified Boyden chambers with polycarbonate membranes containing Matrigel and cultured for 1
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12 h. Cells were then fixed, stained with Gisema soluation, and photographed (×200).
Upper panel: Images show cells that invaded through the pores onto the lower side of the filter. Lower panel: The invading cells were counted in eight randomly chosen microscope fields. Data are shown as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01. (B) Reduced Slug and fascin expression, but increased claudin levels, as determined by western blot analysis, in MDA-MB-231 cells expressing plemiR-30a or Slug, as compared with controls plemiR or shRNA-Luc, respectively. In addition, Slug and fascin protein levels were restored in plemiR-30a–expressing/MDA-MB-231 cells transfected with anti-miR-30a. β-actin was the loading control. (C) Knockdown of Slug notably decreases the invasion of MDA-MB-231 cells, whereas overexpression of miR-30a fails to abrogate the reduced frequency of invasion for sh-Slug/MDA-MB-231 cells. (D) The expression of Slug, fascin, and claudins was also analyzed by western blotting. β-actin was the loading control.
Figure 5: Ectopic expression of miR-30a inhibits tumor growth and metastatic lung colonization of breast cancer xenografts. (A) Representative lungs and hematoxylin and eosin (H&E) staining of metastatic tumor (M) and normal (N) lung tissues from mice 5 weeks after tail vein injection of MDA-MB-231 cells
overexpressing miR-30a or plemiR vector (control). (B) Number of metastatic nodules 1
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in lungs of mice (n = 5 per group) as in (A). (C) Reduced tumor volumes in fat pads of nude mice injected with MDA-MB-231 cells stably overexpressing miR-30a or the control plemiR. The red arrows indicate tumors. (D) Xenograft tumor volumes from mice as in (C). Data represent the mean ± SD. ***P < 0.001. (E) Photographs of representative mice at 5 weeks post-xenotransplantation. Tumors were excised and sectioned and are shown with HE staining and specific staining for expression of Slug, fascin, and claudins, including CLDN-1, -2, and -3. Scale bar = 25 μm.
Figure 6: Correlation between miR-30a and EMT markers. The Pearson correlation coefficient was used to analyze mRNA expression data from 86 patients with breast cancer. (A) Positive correlation between miR-30a and epithelial marker gene CLDN2.
(B) Inverse correlation between miR-30a and mesenchymal marker gene FSCN (C) Inverse correlation between CLDN2 and FSCN. The relative expression of each mRNA was calculated using the comparative CT method as in Materials and methods.
Figure 7: miR-30a inhibits EMT by targeting Slug. (A) The cellular phenotype of miR-30alow/Slughigh/Fascinhigh/Claudinlow correlates with poor clinicopathological features in breast cancer. Immunostaining results for paraffin-embedded breast cancer tissue samples of negative early-stage tumors (stage I; columns a and b) and LNM-positive and advanced-stage tumors (stages III and IV; columns c and d). HE denotes 1
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hematoxylin and eosin staining. In addition, the T/N ratio for miR-30a was <0.50 in columns c and d and was ≥0.50 in columns a and b. Clinicopathological features of the tumors were determined according to the sixth edition of the AJCC Cancer Staging Manual . (B) Schematic representation of the tumor-suppressor role of miR-30a relative to its inhibition of EMT. The targeting of Slug mRNA by miR-30 results in
downregulation of fascin and upregulation of the tight junction proteins CLDN-1, CLDN-2, and CLDN-3, which leads to downregulation of EMT and, ultimately, to a reduced rate of breast cancer progression.
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