Enhanced motility and proliferation by miR-10b/FUT8/p-AKT axis in breast cancer cells

Abstract

Upregulation of microRNA (miR)-10b has been confirmed in multiple types of cancer, however, the role of miR-10b in glycosylation remains unclear. Protein core-fucosylation is an important N-linked glycosylation modification and serves important roles in cancer progression. In a previous study, a glycogene array was applied to profile the alterations of glycogene expression in miR-10b-overexpressed MCF10A cells. Notably, fucosyltranferase 8 (FUT8), which is responsible for the addition of core-fucose to N-glycan, was significantly upregulated by miR-10b. In the present study, increased motility and proliferation were observed in miR-10b-overexpressed MCF10A cells. To assess the mechanism involved, the role of FUT8 in MCF10A cells was studied and it was confirmed that miR-10b promotes motility and proliferation by regulating FUT8 and activating the protein kinase B (AKT) signaling pathway. Consistent with the aforementioned result, decreased motility and proliferation were detected when miR-10b expression was inhibited in MDA-MB-231 cells, transforming growth factor-β-induced and Twist-overexpressed MCF10A cells. To conclude, the findings from the present study indicate that miR-10b promotes motility and proliferation by increasing FUT8 and activating AKT in breast cancer cells.

Keywords: breast cancer; fucosyltranferase 8; microRNA-10b; motility; phosphorylated protein kinase B; proliferation.

Figure 1.

miR-10b promotes the motility and proliferation of BC cells via activation of AKT. (A) Fold-changes of miR-10b were assessed in miR-10b transiently overexpressed MCF10A cells. (B) MCF10A cells transiently transfected with miR-10b and treated with or without MK2206 were subjected to wound healing assay (Scale bar, 200 µm) and transwell assay (×100 magnification). (C) Analysis of proliferation of miR-10b transiently overexpressed MCF10A cells cultured ± MK2206 inhibitor. (D) Western blot analysis of E-cadherin, N-cadherin, vimentin, p-AKT and AKT in miR-10b transiently overexpressed MCF10A cells. (E) Western blot analysis of p-AKT and AKT in miR-10b transiently overexpressed MCF10A cells ± MK2206 inhibitor. **P<0.01, ***P<0.001 vs. NC. BC, breast cancer; AKT, protein kinase B; p-AKT, phosphorylated protein kinase B; miR, microRNA; NC, negative control; E, epithelial; N, neural.

Figure 2.

Effects of FUT8 on cell motility and proliferation. (A) Western blot analysis of EMT markers, p-AKT and AKT, detected in FUT8-overexpressed MCF10A cells. (B) FUT8-overexpressed MCF10A cells were subjected to wound healing and Transwell assay. *P<0.05, ***P<0.001 vs. Mock. (C) Proliferation analysis of FUT8-overexpressed MCF10A cells. (D) Western blot analysis of p-AKT, AKT and FUT8 expression in MCF10A and MDA-MB-231 cells. (E) Western blot analysis of p-AKT, AKT and FUT8 in FUT8-knockdown MDA-MB-231 cells. (F) Wound healing and Transwell assay (Scale bar, 200 µm; ×100 magnification) in FUT8-knockdown MDA-MB-231 cells. (G) MTT was performed in FUT8-knockdown MDA-MB-231 cells. ***P<0.001 vs. NC. FUT8, fucosyltranferase 8; p-AKT, phosphorylated protein kinase B; AKT, protein kinase B; NC, negative control.

Figure 3.

Effects of miR-10b on p-AKT expression in TGF-β-treated and Twist-overexpressed MCF10A cells. (A) miR-10b expression was detected in TGF-β-treated and Twist-overexpressed MCF10A cells and presented as fold-changes. **P<0.01, ***P<0.001 vs. MCF10A. (B) Western blot analysis of FUT8, p-AKT and AKT, detected in Twist-overexpressed MCF10A cells. (C) TGF-β-treated and Twist-overexpressed MCF10A cells were subjected to wound healing and transwell assay ± inhibitor of miR-10b. **P<0.01; ***P<0.001 vs. TGF-β-treated or Twist-overexpressed MCF10A cells. (D) Proliferation analysis in TGF-β-treated and Twist-overexpressed MCF10A cells ± inhibitor of miR-10b. (E) Western blot analysis of FUT8, p-AKT and AKT protein in Twist-overexpressed MCF10A cells, ± inhibitor of miR-10b. (F) Western blot analysis of EMT markers, FUT8, p-AKT and AKT, detected in TGF-β-treated MCF10A cells, ± inhibitor of miR-10b. (Scale bar, 200 µm; ×100 magnification). p-AKT, phospho- protein kinase B; TGF-β, transforming growth factor-β; FUT8, fucosyltranferase; p-AKT, phosphorylated protein kinase B; AKT, protein kinase B; EMT, epithelial-mesenchymal transition; miR, microRNA.

Figure 4.

Inhibitor of miR-10b significantly attenuated the expression of p-AKT and reduced migration and proliferation in MDA-MB-231 cells. (A) miR-10b expression were detected in MDA-MB-231 cells with addition of miR-10b inhibitor. ***P<0.001 vs. MCF10A. (B) Western blot analysis. Expression of FUT8, p-AKT and AKT in MDA-MB-231cells with miR-10b inhibitor. (C) MDA-MB-231 cells were subjected to wound healing and transwell assay (Scale bar, 200 µm; ×100 magnification) ± inhibitor of miR-10b. **P<0.01; ***P<0.001 vs. NC. (D) Analysis of proliferation in MDA-MB-231 cells with addition of miR-10b inhibitor. FUT8, fucosyltranferase; p-AKT, phosphorylated protein kinase B; miR, microRNA; NC, negative control.