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. 2016 Aug 1;6(8):1785-98.
eCollection 2016.

MiR-182 promotes proliferation and invasion and elevates the HIF-1α-VEGF-A axis in breast cancer cells by targeting FBXW7

Chi-Hsiang Chiang  1 , Pei-Yi Chu  2 , Ming-Feng Hou  3 , Wen-Chun Hung  4
Affiliations

MiR-182 promotes proliferation and invasion and elevates the HIF-1α-VEGF-A axis in breast cancer cells by targeting FBXW7

Chi-Hsiang Chiang et al. Am J Cancer Res. .

Abstract

The feature of imperfect complementary effect of miRNAs to mRNAs implies that miRNAs may simultaneously target different mRNAs to affect multiple aspects of tumorigenesis. In our previous results, we demonstrated that miR-182 was over-expressed in breast cancer cell lines and clinical tumor tissues and its up-regulation increased tumorigenicity and invasiveness by repressing a tumor suppressor RECK. In this study, we showed that overexpression miR-182 regulated actin distribution and filopodia formation to increase invasiveness of breast cancer cells. In addition, miR-182 enhanced cell cycle progression and proliferation. We further identified the E3 ubiquitin-protein ligase FBXW7 as a target gene of miR-182. We also demonstrated that miR-182-overexpressing cells were highly sensitive to hypoxia. Under hypoxic condition, HIF-1α and VEGF-A proteins were significantly upregulated in these cells. In addition, the conditioned medium of miR-182-overexpressing cells contained more VEGF-A than the control cells and induced angiogenesis more efficiently in vitro. All these effects could be counteracted by ectopic expression of FBXW7 in cells or neutralization of VEGF-A in the conditioned media by specific antibody. Finally, our data showed that miR-182 expression was inversely correlated with FBXW7 in breast tumor tissues. In conclusion, our study explores a novel mechanism by which miR-182 elevates HIF-1α expression to promote breast cancer progression.

Keywords: FBXW7; HIF-1α; MiR-182; VEGF-A; breast cancer.

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Figures

Figure 1
Figure 1
Overexpression of miR-182 affected the morphology and actin distribution of breast cancer cells. A. The morphology of H184B5F5/M10 and H184B5F5/M10-miR-182 cells. B. The morphology of MCF-7 and MCF7-Sponge cells. C. Immunofluorescent staining showed the alteration of cytoskeleton structure in H184B5F5/M10-miR-182 cells with enriched filopodia. D. The change of cytoskeleton structure in MCF-7 sponge cells compared to MCF-7 cells. E. H184B5F5/M10-miR-182 cells exhibited higher invasive ability than the parental H184B5F5/M10 cells. F. MCF7-Sponge cells showed reduced invasiveness than MCF-7 cells. **P<0.01.
Figure 2
Figure 2
Overexpression of miR-182 increased cell cycle progression, proliferation and clonogenicity of breast cancer cells. A. Overexpression of miR-182 significantly increased the population of cells at the S and G2/M phase in H184B5F5/M10-miR-182 cells. B. Proliferation was significantly increased in H184B5F5/M10-miR-182 cells. C. H184B5/F5/M10-miR-182 cells exhibited higher clonogenicity than the parental H184B5F5/M10 cells. D. Knockdown of miR-182 reduced the population of cells at the S and G2/M phase in MCF-7 cells. E. Kncodown of miR-182 decreased the growth of MCF-7 cells. F. MCF7-Sponge cells showed reduced clonogenicity than that of MCF-7 cells. **P<0.01 and *P<0.05.
Figure 3
Figure 3
FBXW7 was targeted by miR-182. A. FBXW7 mRNA and protein levels were detected in normal mammary epithelial cells and different breast cancer cell lines. B. Overexpression or knockdown of miR-182 could inhibit or increase the protein level of FBXW7 but not the mRNA level. C. The miR-182 targeting sequence in the FBXW7 3’UTR was shown and mutagenesis was performed to change the three nucleotides underlined (upper panel). Wild type or mutated FBXW7 3’UTR reporters were transfected into H184B5F5/M10 and H184B5F5/M10-miR-182 cells and the reporter activities were compared (left panel). In addition, the control reporter vector (pEZX) or FBXW7 3’UTR reporter vector was transfected into H184B5F5/M10-miR-182 cells and the reporter activities were compared (right panel). D. The control reporter vector (pEZX) or FBXW7 3’UTR reporter vector was transfected into MCF-7 or MCF-7 Sponge cells and the reporter activities were compared. ***P<0.001, **P<0.01 and *P<0.05.
Figure 4
Figure 4
MiR-182 affected the protein level of two FBXW7 degradation substrates cyclin E and Notch. A. The protein level of FBXW7, cyclin E and Notch were investigated in different cell lines. B. Knockdown of FBXW7 by siRNA (FBSi) in H184B5F5/M10 cells or ectopic expression of FBXW7 (FBXW7 expression vector, FBEX) in MCF-7 cells modulated the protein level of cyclin E and Notch.
Figure 5
Figure 5
MiR-182 enhanced hypoxia-induced HIF-1α expression and angiogenesis. A. H184B5F5/M10 and H184B5F5/M10-miR-182 cells were cultured in normoxia and 1% O2 for 24 h and the protein level of FBXW7 and HIF-1α was investigated by western blot analysis. B. H184B5F5/M10, H184B5F5/M10-miR-182 and H184B5F5/M10-miR-182 cells transfected with FBXW7 (182-FBXW7) were cultured in normoxia and 1% O2 for 24 h. Total RNAs were isolated and the expression of VEGF-A, -C, and -D was studied by real-time RT-PCR. C. Cells were treated as described in B and the protein level of HIF-1α, FBXW7 and secreted VEGF-A was detected by Western blot analysis. D. H184B5F5/M10 and H184B5F5/M10-miR-182 cells were cultured in 1% O2 for 24 h. The conditioned media were collected and were used to treat endothelial cells (lane 1 and 2). The conditioned media of hypoxia-treated H184B5F5/M10-miR-182 cells were also incubated with control IgG or anti-VEGF-A antibody before treating endothelial cells (lane 3 and 4). The conditioned media of H184B5F5/M10-miR-182 cells transfected with FBXW7 expression vector were collected to treat endothelial cells (lane 5). Results from three independent assays were expressed as Mean ± SE. **P<0.01 and *P<0.05.
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