Hypoxia inhibits the expression of semaphorin 3A in breast cancer cells and regulates the differentiation of preosteoblast cells / 肿瘤

ABSTRACT

Objective: To compare the effect of MCF-7 conditioned medium under normoxia or hypoxia condition on the differentiation of osteoblastic precursor MC3T3-E1 cells after exposing breast cancer MCF-7 cells to hypoxia, and to investigate the role of semaphorin 3A (Sema3A) on osteogenic differentiation regulated by hypoxia in breast cancer with bone metastasis. Methods: The conditioned medium was collected from breast cancer MCF-7 cells cultured under the normoxia or hypoxia condition for 48 h, and used to incubate osteoblastic precursor MC3T3-E1 cells. The content and activity of alkaline phosphatase (ALP) in MC3T3-E1 cells were evaluated by BCIP/NBT ALP color development kit and ALP activity assay kit, respectively. The mineralization of MC3T3-E1 cells was detected by alizarin red S (ARS) staining. The expressions of Sema3A mRNA and protein in MCF-7 cells under normoxia or hypoxia condition for 0, 12, 24 and 48 h were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The recombinant human Sema3A (rhSema3A) was added into MC3T3-E1 cells incubated with MCF-7 hypoxia conditioned medium, then the mRNA expression levels of osteogenic differentiation-related proteins ALP, Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and Osterix (Osx) in MC3T3-E1 cells were measured by real-time fluorescent quantitative PCR. Results: Compared with normoxia cultured MCF-7 conditioned medium (NorCM) group, the activity of ALP in MC3T3-E1 cells was significantly decreased in hypoxia cultured MCF-7 conditioned medium (HyCM) group (P<0.05), and the area of mineralization nodes stained with ARS was also markedly reduced in HyCM group (P<0.05). The mRNA and protein expressions of Sema3A in MCF-7 cells after hypoxia culture for 24 and 48 h were significantly lower than those after normoxia culture for the same time (all P<0.05). After rhSema3A was used to treat MC3T3-E1 cells which were incubated with HyCM, the mRNA expression levels of ALP, RUNX2, OCN and Osx were significantly increased as compared with the rhSema3A untreated group (all P<0.05). Conclusion: Hypoxia down-regulates the expression of Sema3A in MCF-7 cells in vitro, and inhibits the differentiation of osteoblast, which indicates that Sema3A is probably an important factor for the inhibition of osteoblastic differentiation by breast cancer conditioned medium under hypoxic environment.