miR-137 alleviates doxorubicin resistance in breast cancer through inhibition of epithelial-mesenchymal transition by targeting DUSP4

Abstract

Acquired resistance to chemotherapy is a major obstacle in breast cancer (BC) treatment. Accumulated evidence has uncovered that microRNAs (miRNAs) are vital regulators of chemoresistance in cancer. Growing studies reveal that miR-137 acts as a suppressor in tumor progression. However, it remains obscure the role of miR-137 in modulating the sensitivity of BC cells to doxorubicin (DOX). In this study, we demonstrate that miR-137 exerts a significant effect on repressing the development of chemoresistance of BC cells in response to DOX via attenuating epithelial-mesenchymal transition (EMT) of tumor cells in vitro and in vivo. MiR-137 overexpression dramatically elevated the sensitivity of BC cells to DOX as well as impaired the DOX-promoted EMT of tumor cells. Mechanistically, miR-137 directly targeted dual-specificity phosphatase 4 (DUSP4) to impact on the EMT and chemoresistance of BC cells upon DOX treatment. Consistently, decreased DUSP4 efficiently enhanced the sensitivity of BC cells to DOX while overexpressed DUSP4 significantly diminished the beneficial effect of miR-137 on BC cells chemoresistance. Moreover, the increased miR-137 heightened the sensitivity of BC cells-derived tumors to DOX through targeting DUSP4 in vivo. Together, our results provide a novel insight into the DOX resistance of BC cells and miR-137 may serve as a new promising therapeutic target for overcoming chemoresistance in BC.

Fig. 1. miR-137 overexpression sensitized BC cells to DOX.

a CCK-8 showing viability of BC cell lines under DOX treatment (0, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20 μg/ml) for 48 h; the IC50 value was calculated based on the CCK-8 results. b Detection of miR-137 expression in BC cells; the result was quantified by comparing with the internal control U6. c–g Detection of viability of BC cells transfected with miR-137 mimics (5 nM) or negative control (NC) RNA and cultured with 0, 0.5, 1.0, 1.5, and 2.0 µg/ml DOX for 48 h. h–l Detection of viability of BC cells transfected with miR-137 inhibitor (5 nM) or NC RNA and cultured with 0, 0.5, 1.0, 1.5, and 2.0 µg/ml DOX for 48 h. All data are representative of three independent experiments.

Fig. 2. miR-137 overexpression inhibited DOX-induced EMT in BC cells.

a Effect of DOX (IC50) on E-cadherin and vimentin expression in BC cell lines determined by western blotting. b Effect of DOX (IC50) on miR-137 expression in BC cell lines detected by qPCR. c Western blotting detection of E-cadherin and vimentin expression levels in BC cells treated with control, DOX, or DOX plus miR-137 mimics. d Immunofluorescence detection of E-cadherin and vimentin expression in BC cells treated with control, DOX, or DOX plus miR-137 mimics. *P < 0.05 versus control. All data are representative of three independent experiments.

Fig. 3. miR-137 regulated DUSP4 negatively.

a TargetScan-predicted binding sequences of miR-137 in the 3′-UTR of DUSP4. b Quantification of DUSP4 mRNA expression in BC cells transfected with miR-137 mimics, miR-137 inhibitor, or NC. c, d Quantification of DUSP4 protein expression in BC cells transfected with miR-137 mimics, miR-137 inhibitor, or NC. All data are representative of three independent experiments.

Fig. 4. DUSP4 knockdown inhibited DOX-induced EMT in BC cells.

a DUSP4 protein levels in MCF-7 and MCF-7/ADR cells. b, c Detection of viability of MCF-7 and MCF-7/ADR cells transfected with DUSP4 siRNA or NC and cultured with 0, 0.5, 1.0, 1.5, and 2.0 µg/ml DOX. d Western blotting confirmation of RNA interference efficiency of DUSP4 siRNA. e Western blotting detection of E-cadherin and vimentin expression levels in BC cells treated with control, DOX, or DOX plus DUSP4 siRNA. All data are representative of three independent experiments.

Fig. 5. DUSP4 knockdown eliminated miR-137 inhibitor-mediated regulation of DOX sensitivity and EMT.

a, b CCK-8 detection of viability of MCF-7 and MCF-7/ADR cells transfected with DUSP4 siRNA alone or with both DUSP4 siRNA and miR-137 inhibitor and cultured with 0, 0.5, 1.0, 1.5, and 2.0 µg/ml DOX. c Western blotting detection of E-cadherin and vimentin expression in MCF-7 and MCF-7/ADR cells transfected with DUSP4 siRNA alone or with both DUSP4 siRNA and miR-137 inhibitor. All data are representative of three independent experiments.

Fig. 6. DUSP4 overexpression reversed miR-137 mimic-mediated regulation of DOX sensitivity and EMT.

a, b CCK-8 detection of viability of MCF-7 and MCF-7/ADR cells transfected with DUSP4 vector or with both DUSP4 vector and miR-137 mimic and cultured with 0, 0.5, 1.0, 1.5, and 2.0 µg/ml DOX. c Western blotting detection of E-cadherin and vimentin expression in MCF-7 and MCF-7/ADR cells transfected with DUSP4 vector or with both DUSP4 vector and miR-137 mimic. All data are representative of three independent experiments.

Fig. 7. miR-137 overexpression enhanced the anti-BC activity of DOX in vivo.

a, b Representative tumor images (n = 6 mice per group). c Tumor volumes were measured every other day. The tumor regression rate was calculated based on the tumor volumes. *P < 0.05, **P < 0.01, ***P < 0.001. d The mouse body weight in each group was measured every other day.