MicroRNA-590-3p inhibits invasion and metastasis in triple-negative breast cancer by targeting Slug

Abstract

miR-590-3p acts as a tumor suppressor in glioblastoma multiform, medulloblastoma, hepatocellular carcinoma, and nephroblastoma. Here, we studied the role of miR-590-3p in triple-negative breast cancer (TNBC). The miR-590-3p levels in TNBC specimens were significantly lower than those in non-TNBC specimens. Overexpression of miR-590-3p significantly inhibited migration and invasion of TNBC cells and lung metastasis in vivo. Interestingly, miR-590-3p decreased the Slug mRNA and protein levels in TNBC cells, and luciferase reporter assay showed that miR-590-3p directly targeted 3'-UTR of Slug in TNBC cells. Importantly, overexpression of Slug reversed the inhibitory effect of miR-590-3p on migration and invasion of TNBC cells. Taken together, miR-590-3p inhibits TNBC migration and invasion by directly targeting Slug, suggesting a potential therapeutic effect of miR-590-3p for TNBC.

Keywords: Slug; Triple-negative breast cancer; cancer metastasis; miR-590-3p.

Figure 1

Expression levels of miR-590-3p are down-regulated in breast cancer specimens and cell lines. A. Levels of miR-590-3p in breast cancer cell lines and human mammary epithelial cells, as determined using qRT-PCR. B. Levels of miR-590-3p in TNBC (n = 42) and non-TNBC (n = 18) tumor tissues, as determined using qRT-PCR. C. Frequency of distant organ metastasis (lung, bone and liver) 5 years post-surgery in TNBC patients with the miR-590-3p^high or miR-590-3p^low tumors. Data are presented as mean ± SD from three independent experiments. The differences in expression levels of miR-590-3p between different cell lines or between different tumor tissues were analyzed using one-way ANOVA and followed by the SNK test. Median values are represented by the horizontal line in the middle. *, P < 0.05 was considered statistically significant, denoted by **, P < 0.01; ***, P < 0.001; and ns, no significance.

Figure 2

Overexpression of miR-590-3p inhibits migration and invasion of TNBC cells in vitro and metastasis in vivo. A. Representative images of wound healing assay (left panel) show the migration of MDA-MB-231 and BT-549 cells transfected with miR-590-3p-NC or miR-590-3p mimics, at the beginning (t = 0 h) and the end (t = 24 h) of the recording. The graph (right panel) shows the numbers of the migrated cells transfected with miR-590-3p mimics or miR-590-3p-NC. **, P < 0.01. B. Representative images of transwell assay (left panel) show the invasion of MDA-MB-231 and BT549 cells transfected with miR-590-3p-mimics or miR-590-3p-NC. The graph (right panel) shows the numbers of the invaded cells transfected with miR-590-3p mimics or miR-590-3p-NC. **, P < 0.01. C. Representative IVIS images (left panel) show lung metastases in mice that were injected with luciferase-labeled MDA-MB-231 cells transfected with miR-590-3p mimics or the miR-590-3p-NC control. The graph (right panel) shows quantitation of lung metastases on day 40 after implantation of tumor cells, as assessed by bioluminescence measurements (n = 5). The color scale bar depicts the photon flux (photons per second) emitted from these mice. Data are presented as mean ± SD from three independent experiments. The differences in migration, invasion, and metastasis between the miR-590-3p-NC and the miR-590-3p-mimic cells were analyzed using two-tailed Student’s t-test. **, P < 0.01 was considered statistically significant.

Figure 3

miR-590-3p directly targets Slug, and its expression level is correlated with a high metastasis rate in TNBC patients. A. RNA sequence alignment shows that the 3’-UTR of Slug mRNA contains a complementary site for the seed region of miR-590-3p. Slug-mut, a mutant with substitutions in the complementary region, was used as a negative control. B. Enforced expression of miR-590-3p suppressed the expression of Slug protein in TNBC cell lines, as determined by Western blotting. C. The luciferase activity was determined using dual luciferase reporter assay after transfection of the luciferase reporter vector (psiCHECK-2-Slug-3’UTR-WT or psiCHECK-2-Slug-3’UTR-MUT) into miR-590-3p-mimic or miR-590-3p-NC-transfected MDA-MB-231 cells. The data represent the means ± SD of three independent experiments. The luciferase activity was measured. Data are presented as mean ± SD from three independent experiments; ***, P < 0.001 compared with the negative control; and ns, no significance. D. The mRNA levels of Slug were reversely correlated with the expression levels of miR-590-3p in TNBC tissue specimens (n = 42), as determined by qRT-RCR. The correlation between the mRNA levels of Slug and the expression levels of miR-590-3p in TNBC tumors were analyzed using Pearson test. P < 0.001 was considered statistically significant.

Figure 4

miR-590-3p inhibits the EMT phenotype by suppressing Slug expression in TNBC cells. (A) Expression levels of miR-590-3p and Slug mRNA in MDA-MB-231 and BT-549 cells were determined using qRT-PCR after transfection with miR-590-3p-NC or miR-590-3p mimic along with or without the Slug. (B) Western blots show the protein expression levels of Slug, E-cadherin, and Vimentin in MDA-MB-231 and BT-549 cells, which were transfected with miR-590-3p-NC or miR-590-3p mimic along with or without the Slug. β-actin was used as a loading control. (C) Representative images (left panel) show the migration of BT-549 and MDA-MB-231 cells after transfection with miR-590-3p-NC or miR-590-3p mimic along with or without the Slug at 0 h and 24 h of recording of migration. The graph (right panel) shows that the inhibitory effect of miR-590-3p on cell migration was largely reversed by Slug overexpression. **, P < 0.01. (D) Representative images show the invasion of MDA-MB-231 and BT549 cells after transfection with miR-590-3p-NC or miR-590-3p mimic along with or without the Slug. The graph (right-lower panel) shows that the inhibitory effect of miR-590-3p on cell invasion was largely reversed by Slug overexpression. **, P < 0.01. In (C and D), differences in migration and invasion among cells transfected with miR-590-3p-NC or miR-590-3p mimic along with or without the Slug. were analyzed using two-tailed Student’s t-test. **, P < 0.01 was considered statistically significant.