Lipid raft-mediated miR-3908 inhibition of migration of breast cancer cell line MCF-7 by regulating the interactions between AdipoR1 and Flotillin-1

Figures

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  Fig. 1. Expression of AdipoR1 was suppressed by miR-3908 in MCF-7 cells. To study if microRNA modulates AdipoR1, or the downstream pathway of AdipoR1 in MCF-7 cells, firstly, using the online software Targetscan (http://targetscan.org/), we tried to forecast miRNAs that target AdipoR1. Within these miRNAs, miR-3908 potentially co-targeted mRNAs of AdipoR1 3’-UTRs. A further proof experiment was carried out with a luciferase reporter gene assay to identify whether 3’-UTRs of AdipoR1 were bonded directly with miR-3908. The results showed that the relative luciferase activities of AdipoR1 3’-UTR were obviously downregulated in MCF-7 cells transfected with miR-3908 (a), but there was no change in the mut-AdipoR1 group (b). However, it was identified that miR-3908 could not form direct bonds with AMPK (c), SIRT-1 (d), or Flotillin-1 (e) 3’-UTRs, respectively. The data are presented as means ± SD from three independent experiments. *P < 0.05, **P < 0.01

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  Fig. 2. Expression of AdipoR1 was suppressed by miR-3908 in MCF-7 cells. To verify the miR-3908 effect on the mRNA and protein expression of AdipoR1, AMPK, SIRT-1, and Flotillin-1 in MCF-7 cells, we accomplished relative quantification analyses with qRT-PCR and Western blots, respectively. miR-3908 inhibited the mRNA expression of AdipoR1 in MCF-7 cells (a), as well as the protein expression of AdipoR1 (b). Moreover, miR-3908 did not affect the mRNA expression of AMPK, SIRT-1, and Flotillin-1, but the protein expression of AMPK, p-AMPK, SIRT-1, and Flotillin-1 also decreased (b). Furthermore, the inhibitors of miR-3908 promoted the mRNA expression of AdipoR1 in MCF-7 cells, but did not affect the mRNA expression of AMPK, SIRT-1, and Flotillin-1 (a). Altogether, our results demonstrated that miR-3908 directly regulates the expression of AdipoR1 by interacting with its 3’-UTR, and then the protein expression of its downstream genes. The data are presented as means ± SD from three independent experiments. *P < 0.05, **P < 0.01

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  Fig. 3. miR-3908 suppresses cell growth and clonogenicity in MCF-7 cells. For evaluating the overexpressed or underexpressed possible biotic outcomes induced by miR-3908, several functional experiments were performed in MCF-7 cells. Compared with control cells, proliferation of MCF-7 cells (a) would be suppressed by transfection with miR-3908. Moreover, the inhibitors of miR-3908 accentuated the same process in MCF-7 cells (a). Furthermore, the colony formation assay revealed that miR-3908 played a significant role in the suppression of this ability compared with the control group in MCF-7 cells. However, downregulation of miR-3908 facilitated significantly the colony-forming ability of MCF-7 cells. These results suggested that miR-3908 also lowered colony formation of MCF-7 cells (b) (P < 0.001). The data are presented as means ± SD from three independent experiments. *P < 0.05, **P < 0.01

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  Fig. 4. miR-3908 suppresses migration of MCF-7 cells. miR-3908 inhibited significantly the migration ability of MCF-7 cells compared to control (NC) (a and b) in vitro (P < 0.001). Conversely, using the inhibitors of miR-3908, the migration ability was enhanced in MCF-7 cells (a and b). These results suggested that miR-3908 suppresses the migration of MCF-7 cells. The data are presented as means ± SD from three independent experiments. *P < 0.05, **P < 0.01

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  Fig. 5. AdipoR1 protein interacted with Flotillin-1. The proteins that interacted with AdipoR1 were analyzed by co-immunoprecipitation (CO-IP) and confocal laser scanning microscopy (CLSM). MCF-7 cells were harvested after 48 h in culture, then total cellular proteins were extracted and CO-IP’d (co-immunoprecipitated) with anti-AdipoR1 antibodies followed by anti-Flotillin-1 antibodies (a). Then, the above proteins were then Co-IP’d with anti-Flotillin-1 antibodies followed by AdipoR1 antibodies (a). AdipoR1 is localized to the cell membrane of EC. After MCF-7 cells were co-transfection with plasmids pEGFP-C1-Flotillin-1 (green) and pDS-RED1-N1-AdipoR1 (red) for 48 h, the confocal scanning images demonstrated that the Flotillin-1 recombinant protein was localization to the cell membrane. The nuclei of the cells (blue) were stained by DAPI. The overlaid image indicates that AdipoR1 overlapped with Flotillin-1 at the MCF-7 cell membrane (b)

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  Fig. 6. The inhibitors of miR-3908 induced the mobility of AdipoR1 into lipid rafts. In the last few years, many researches have indicated that functional receptors assemble at the platforms provided by lipid rafts. In metastasis and progression of breast cancer (BC), AdipoR1 plays an important role, and may be a potential alternative therapeutic goal for patients with BC. The migration of AdipoR1 into lipid rafts plays an important role in the anti-invasion process. To explore the anti-invasion functions of miR-3908 inhibitors, the role of these inhibitors on the mobility of AdipoR1 into lipid rafts was determined. Our results demonstrated that miR-3908 inhibitors reduced the migration of AdipoR1 into fractions of the lipid raft. The phenomenon could be enhanced by miR-3908, but reversed by inhibitors of miR-3908 as well as by Flotillin-1