Subjects: Medicine, Pharmacy >> Preclinical Medicine submitted time 2017-12-27 Cooperative journals: 《南方医科大学学报》
Abstract： Objective To investigate the effect of precursor of nerve growth factor (proNGF) in promoting invasion of breast cancer cells and its relation with ezrin expression and phosphorylation of ezrin Thr567 and Tyr477. Methods Human breast cancer cell lines MDA-MB-231 and MCF-7 were stimulated by gradient concentrations of proNGF (0, 2.5, 5 and 10 ng/mL) for 16 h, and the invasion of the cells was assessed with Transwell assay. The expression of ezrin and the phosphorylation of ezrin Thr567 and ezrin Tyr477 in the treated cells were examined by Western blotting. MDA-MB-231 cells were transfected with pEnter-His-ezrinY477F (a dominant negative mutant) to study the role of phosphrylation of ezrin Tyr477 in the invasion of breast cancer cell stimulated by proNGF. Results proNGF significantly promoted MDA-MB-231 and MCF-7 cell invasion in a concentration-dependent manner (P<0.05), and concentration- and time-dependently increased the phosphorylation of ezrin Tyr477 (P<0.05) without affecting the expression of ezrin or the phosphorylation of ezrin Thr567. The specific inhibitor of src, SKI-606, significantly inhibited the phosphorylation of ezrin Tyr477 induced by proNGF. Transfection with pEnter-HisezrinY477F inhibited proNGF-induced invasion and phosphorylation of ezrin Tyr477 in MDA-MB-231 cells (P<0.05). Conclusion Phosphorylation of ezrin Tyr477 plays a critical role in the invasion of breast cancer cells stimulated by proNGF via proNGF/src/ezrin Tyr477 pathway.
Subjects: Medicine, Pharmacy >> Preclinical Medicine submitted time 2017-12-27 Cooperative journals: 《南方医科大学学报》
Abstract： Objective To study the relationship between Nanog-promoted metastasis of breast cancer and ezrinT567 phosphorylation, and explore the possible mechanism by which Nanog regulates ezrinT567 phosphorylation. Methods A siRNA construct targeting Nanog was transfected in breast cancer cells to knock down Nanog expression, and the changes in the cell invasion was detected using Transwell assay. The expression levels of Nanog and PKC and the phosphorylation level of ezrinT567 were detected using Western blotting and immunofluorescent staining; the protein interaction between PKCɛ and ezrin was assayed by co-immunoprecipitation and Western blotting. Results Nanog knockdown significantly decreased the expression of PKCɛ protein, phosphorylation level of ezrinT567 and the invasion ability of breast cancer cells. PKCε knockdown obviously decreased the phosphorylation level of ezrinT567 in the cells, and PKCɛ and ezrin were co-immunoprecipitated. Concludions Nanogcan can upregulate the expression of PKCɛ to promote the phosphorylation of ezrinT567, which can be a new mechanism by which Nanog promotes tumor metastasis.
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