miR-3178 inhibits cell proliferation and metastasis by targeting Notch1 in triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) has a poorer outcome than other subtypes of breast cancer, and the discovery of dysregulated microRNA (miRNA) and their role in tumor progression has provided a new avenue for elucidating the mechanism involved in TNBC. In this study, we identified that miR-3178 was significantly reduced in TNBC, and the low miR-3178 expression correlated with poor overall survival in TNBC but not in non-TNBC. The ectopic overexpression of miR-3178 suppressed TNBC cell proliferation, invasion, and migration by inhibiting the epithelial-to-mesenchymal (EMT) transition. Notch1 was validated as the direct target gene of miR-3178, which was confirmed by the dual-luciferase reporter assay. miR-3178 decreased the expression of Notch1 and restoration of Notch1 expression attenuated the inhibitory effects of miR-3178 on cell proliferation, metastasis, and the EMT in TNBC. miR-3178 inhibited cell proliferation and metastasis by targeting Notch1 in TNBC, and the restoration of miR-3178 might be a potential therapeutic strategy for TNBC.

Fig. 1. miR-3178 expression level was downregulated in TNBC.

a The strategy for detecting different expression miRNAs between TNBC and non-TNBC in TCGA database. b Scatter plot of the different expression miRNAs. The points under the red line represent the differentially expressed miRNAs with statistical significance. c Expression levels of miR-3178 in TNBC and non-TNBC in TCGA. d Expression levels of miR-3178 in 17 TNBC and 17 non-TNBC tissues. e, f High levels of miR-3178 correlated with a better overall survival in TNBC. However, high miR-3178 expression was not associated with a better OS in non-TNBC

Fig. 2. miR-3178 suppressed the cell proliferation and migration of TNBC cells.

a Cells were transfected with miR-3178 mimic lentivirus, and the expression level of miR-3178 was detected after stable transfection. b Cell proliferation was evaluated by the CCK-8 assay at 24, 48, 72, and 96 h. c The effect of miR-3178 on mRNA expression levels of cell proliferation and cycle-related genes was detected by qRT-PCR. d Colony formation assay showed miR-3178 reduced colony numbers. The wound healing assay (e) and transwell invasion assay (f) were performed to detect the effects of miR-3178 on TNBC migration and invasion, scale bars: 300 μm (e) and 100 μm (f). ***P < 0.001, *P < 0.05

Fig. 3. miR-3178 inhibited the EMT in TNBC cells.

qRT-PCR (a) and western blot (b) analysis showed miR-3178 increased E-cadherin levels, and inhibited vimentin and N-cadherin levels in SUM-1315 and MDA-MB-231. c miR-3178 downregulated the mRNA expression levels of Snail and Slug, transcription factors that directly regulate the EMT. d Snail and Slug protein expression in SUM-1315 and MDA-MB-231 transfected with miR-3178 mimics. *P < 0.05

Fig. 4. Notch1 was a direct downstream target of miR-3178.

a The luciferase reporter constructs that had either a WT-notch1-3′-UTR or mut-notch1-3′-UTR sequence of the miR-3178-binding site. b Luciferase reporter activity revealed miR-3178 suppressed Notch1 3′UTR luciferase activity of wide-type constructs in SUM-1315 and MDA-MB-231 cells. c qRT-PCR analysis showed that miR-3178 mimics decrease Notch1 and NF-κB (a downstream factor of Notch1) mRNA level. d Notch1 and NF-κB protein expression in SUM-1315 and MDA-MB-231 transfected with miR-3178 mimics. * P < 0.05

Fig. 5. Expression of Notch1 attenuated inhibition of cancer cell proliferation by miR-3178 in TNBC cells.

Cells were transfected with Notch1 overexpression plasmid (p-Notch1), and the mRNA and protein expression of Notch1 and NF-κB protein level was detected by qRT-PCT (a) and western blot (b). c CCK8 assay showed the inhibition of cell proliferation by miR-3178 was reversed by p-Notch1. d p-Notch1restoredthe mRNA levels of Ki-67, cyclinD1, and cyclin E1, which were inhibited by miR-3178 mimics. e p-Notch1 reversed the inhibitory effects of miR-3178 on colony formation. **P < 0.01, *P < 0.05

Fig. 6. miR-3178 regulated Notch1-mediated migration and the EMT through Snail1 in TNBC cells.

The wound healing (a) and transwell assay (b) showed the inhibition of miR-3178 on cell migration and invasion was attenuated after restoring the expression of Notch1 by p-Notch1, scale bars: 300 μm (a) and 100 μm (b). c p-Notch1 reversed the effect of miR-3178 on the mRNA expression of the EMT markers and Snail. d p-Notch1 restored the protein expression of the EMT markers and Snail. *** P <0.001, ** P <0.01, * P < 0.05

Fig. 7. miR-3178 inhibited tumor growth in a nude mouse model of TNBC.

a MDA-MB-231 and SUM-1315 cells transfected with miR-3178, miR-3178/p-Notch1, miR-3178/p-NC, and m-NC were subcutaneously implanted into nude mice. The tumor weight of miR-3178 and miR-3178/p-NC was lower than that with m-NC and miR-3178/p-Notch1. b miR-3178 significantly decreased tumor volume compared with m-NC. miR-3178/p-Notch1 partly neutralized the inhibitory effects of miR-3178 on tumor volume. ***P < 0.001, **P < 0.01, *P < 0.05