Upregulation and biological function of transmembrane protein 119 in osteosarcoma

Abstract

Osteosarcoma is suggested to be caused by genetic and molecular alterations that disrupt osteoblast differentiation. Recent studies have reported that transmembrane protein 119 (TMEM119) contributes to osteoblast differentiation and bone development. However, the level of TMEM119 expression and its roles in osteosarcoma have not yet been elucidated. In the present study, TMEM119 mRNA and protein expression was found to be up-regulated in osteosarcoma compared with normal bone cyst tissues. The level of TMEM119 protein expression was strongly associated with tumor size, clinical stage, distant metastasis and overall survival time. Moreover, gene set enrichment analysis (GSEA) of the Gene Expression Omnibus (GEO) GSE42352 dataset revealed TMEM119 expression in osteosarcoma tissues to be positively correlated with cell cycle, apoptosis, metastasis and TGF-β signaling. We then knocked down TMEM119 expression in U2OS and MG63 cells using small interfering RNA, which revealed that downregulation of TMEM119 could inhibit the proliferation of osteosarcoma cells by inducing cell cycle arrest in G0/G1 phase and apoptosis. We also found that TMEM119 knockdown significantly inhibited cell migration and invasion, and decreased the expression of TGF-β pathway-related factors (BMP2, BMP7 and TGF-β). TGF-β application rescued the inhibitory effects of TMEM119 knockdown on osteosarcoma cell migration and invasion. Further in vitro experiments with a TGF-β inhibitor (SB431542) or BMP inhibitor (dorsomorphin) suggested that TMEM119 significantly promotes cell migration and invasion, partly through TGF-β/BMP signaling. In conclusion, our data support the notion that TMEM119 contributes to the proliferation, migration and invasion of osteosarcoma cells, and functions as an oncogene in osteosarcoma.

Figure 1

TMEM119 overexpression is associated with poor survival of osteosarcoma patients. (a) TMEM119 mRNA expression was significantly higher in osteosarcoma tissues (n=81) than in normal osteoblasts (n=6) from GSE42352 (P<0.05). (b) The level of TMEM119 mRNA was significantly elevated in osteosarcoma tissues (n=45) compared to that in bone cyst tissues (n=20) from patients admitted to Affiliated Yixing Hospital of Jiangsu University and the First Affiliated Hospital of Soochow University (P<0.0001). (c) TMEM119 protein expression was determined by western blotting analysis in osteosarcoma (T1–T4) and bone cyst tissues (N1–N4). The experiments were repeated at least three times with similar results. (d) TMEM119 protein expression in osteosarcoma tissues was determined by IHC staining. Magnification: × 200, scale bars: 100 μm. (e) The overall survival time of 100 patients with osteosarcoma based on IHC staining (P=0.0003, HR=0.411, 95% CI: 0.254–0.668). (f) Survival analysis of patients from GSE39055 (P=0.0011, HR=0.118, 95% CI: 0.0.28–0.428).

Figure 2

GESA on the GSE42352 dataset. Enrichment plots of gene expression signatures for cell cycle (a), cell apoptosis (b), metastasis (c) and TGF-β (d) pathways according to TMEM119 expression level. NES, normalized enrichment score.

Figure 3

Suppressing TMEM119 expression represses the proliferation of osteosarcoma cells. (a) TMEM119 expression in five osteosarcoma cell lines was analyzed by real-time PCR (left panel) and western blotting (right panel) using GAPDH as the internal control. The experiments were repeated at least three times with similar results. (b) TMEM119 expression in U2OS and MG63 cells was analyzed by real-time PCR (left panel) and western blotting (right panel). The experiments were repeated at least three times with similar results. (c) Cell proliferation was detected in siRNA-transfected U2OS and MG63 cells by the CCK-8 assay. ***P<0.001.

Figure 4

Suppressing TMEM119 expression induces G0/G1-phase arrest and apoptosis in osteosarcoma cells. (a) U2OS and MG63 cells cultured in 6-well plated were transfected with the indicated siRNA, and the cells were collected 48 h later. Cell cycle distribution was analyzed by PI staining and flow cytometry. (b) Protein levels of cell cycle-related proteins (PCNA, CDC25A and CDK1) in osteosarcoma cell lines were detected by western blotting. (c) Apoptosis was analyzed by Annexin V/PI staining. (d) Levels of apoptosis-related proteins (caspase-9, caspase-8 and Bcl2) were detected by western blotting. The experiments were repeated at least three times with similar results. ***P<0.001.

Figure 5

Knockdown of TMEM119 in osteosarcoma cells suppresses tumor growth in vivo. (a) U2OS cells were subcutaneously injected in athymic nude mice. Tumor volume was measured for 45 days after TMEM119 siRNA or control siRNA injection (n=6). *P<0.05, **P<0.01, ***P<0.001. (b) At day 45, the mice were euthanized, and xenografts were weighed (n=6, P<0.0001). (c) TMEM119 expression in xenografts from nude mice was determined by western blotting. The experiments were repeated at least three times with similar results. (d) Ki67 immunostaining and (e) the TUNEL assay were performed on the xenografts. Magnification: × 200, scale bars: 100 μm.

Figure 6

Silencing of TMEM119 inhibits the migration and invasion of osteosarcoma cells. (a, b) Migration and invasion assays were performed on siRNA-transfected U2OS (a) and MG63 cells (b). Magnification: × 200, scale bars: 100 μm. (c) Protein levels of metastasis-related proteins (MMP2, Twist1, ZEB1, E-cadherin, N-cadherin, vimentin and α-SMA) in osteosarcoma cell lines were detected by western blotting. (d) Protein levels of TGF-β pathway-related factors (BMP2, BMP7 and TGF-β) in U2OS and MG63 cells were detected by western blotting. The experiments were repeated at least three times with similar results. **P<0.01, ***P<0.001.

Figure 7

Exogenous TGF-β rescues the inhibitory effects of TMEM119 siRNA on cell migration and invasion. Cell migration and invasion assays were performed on siRNA-transfected U2OS (a) and MG63 (b) cells in a Boyden chamber containing either DMSO (vehicle) or 10 ng ml⁻¹ TGF-β (Sigma). Magnification: × 200, scale bars: 100 μm. **P<0.01, ***P<0.001.

Figure 8

TGF-β inhibitor or BMP inhibitor impairs the effects of TMEM119 overexpression on migration and invasion. (a, b) Saos2 cells were infected with vector of the TRMEM119 lentivirus, and migration and invasion assays were performed in a Boyden chamber containing either DMSO (vehicle) or 10 μM SB431542 (SB; Sigma). Magnification: × 200, scale bars: 100 μm. (c) Phosphorylation of Smad2/3 was determined by western blotting. (d, e) Saos2 cells infected with vector of the TRMEM119 lentivirus, and migration and invasion assays were performed in a Boyden chamber containing either DMSO (vehicle) or 5 μM dorsomorphin (DM; Sigma). Magnification: × 200, scale bars: 100 μm. (f) Phosphorylation of Smad1/5/8 was determined by western blotting. The experiments were repeated at least three times with similar results. *P<0.05, **P<0.01, ***P<0.001 (n=3).