Lung cancer cell-derived Sema4D mediates osteolytic bone destruction in lung cancer by inhibiting osteoblast differentiation
10.3781/j.issn.1000-7431.2019.11.332
- Author:
Wugui CHEN
1
Author Information
1. Department of Orthopedics, Second Affiliated Hospital, Military Medical University
- Publication Type:Journal Article
- Keywords:
Bone;
Cell differentiation;
Lung neoplasms;
Neoplasm metastasis;
Osteoblasts;
Semaphorin 4D
- From:
Tumor
2019;39(9):701-711
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To determine the role of semaphorin 4D (Sema4D) in osteolytic bone destruction of bone metastasis in lung cancer, and to explore its mechanism. Methods: Immunohistochemistry was used to detect the expression of Sema4D in bone metastases of lung cancer and normal bone tissues. The expression levels of Sema4D protein and mRNA in four kinds of lung cancer cell lines were detected by Western blotting and real-time fluorescent quantitative PCR, respectively. The conditioned medium of lung cancer cells was collected, and ELISA was used to detect the secretion level of Sema4D in lung cancer conditioned medium. The conditioned medium from lung cancer cells with Sema4D high expression (PC9 cells) or low expression (A549 cells) was collected and co-cultured with osteoblastic precursor MC3T3-E1 cells, then the osteoblast differentiation ability was detected by alkaline phosphatase staining, the mineralization ability of osteoblasts was detected by alizarin red staining, the expression levels of osteoblast differentiation genes alkaline phosphatase, liver/bone/kidney (ALPL), Oxterix and collagen type alpha 1 (Col1α1) were detected by real-time fluorescent quantitative PCR. Sema4D shRNA was transfected into lung cancer PC9 cells, then the expression and secretion levels of Sema4D in lung cancer cells were detected by Western blotting, real-time fluorescent quantitative PCR and ELISA, respectively. The conditioned medium of lung cancer PC9 cells transfected with Sema4D shRNA was collected and co-cultured with preosteoblasts, and the effect of the conditioned medium from lung cancer cells with Sema4D interference on the osteoblast differentiation was determined by alkaline phosphatase staining, alizarin red staining and real-time fluorescent quantitative PCR, respectively. Results: Compared with the normal bone tissues, Sema4D was highly expressed in osteolytic bone metastases of lung cancer. Sema4D was expressed and secreted in different lung cancer cell lines in different degrees. The expression and secretion levels of Sema4D were the highest in lung cancer PC9 cells, but lowest in lung cancer A549 cells (P < 0.05). The conditioned medium of lung cancer cells significantly inhibited the osteogenic differentiation of osteoblasts induced by osteogenic inducer, which was characterized by the reduction of relative staining area of alkaline phosphatase staining and alizarin red staining (both P < 0.05), and the decreased expression levels of osteogenic differentiation genes ALPL, Oxterix and Col1α1 (all P < 0.05). The conditioned medium of PC9 cells with Sema4D high expression exhibited more aggressive inhibitory effect on osteoblast differentiation than the conditioned medium of A549 cells with Sema4D low expression (P < 0.05). shRNA transfection significantly reduced the expression and secretion of Sema4D in lung cancer cells (both P < 0.05), and significantly attenuated the inhibitory effect of lung cancer conditioned medium on osteoblast differentiation (all P < 0.05). Conclusion: Lung cancer-derived Sema4D plays an important role in osteolytic bone destruction by inhibiting osteoblast differentiation, suggesting that it is expected to become a new therapeutic target for bone metastasis of lung cancer.
