TRIM66 overexpresssion contributes to osteosarcoma carcinogenesis and indicates poor survival outcome

Abstract

TRIM66 belongs to the family of tripartite motif (TRIM)-containing proteins. Alterations in TRIM proteins have been implicated in several malignancies. This study was aimed at elucidating the expression and biological function of TRIM66 in osteosarcoma. Here, TRIM66 expression level was higher in osteosarcoma tissues than in normal tissues. High TRIM66 expression was correlated with high rate of local recurrence and lung metastasis, and short survival time. Then, we found that knockdown of TRIM66 in two osteosarcoma cell lines, MG63 and HOS, significantly inhibited cell proliferation and induced G1-phase arrest. Moreover, inhibition of TRIM66 in osteosarcoma cells significantly induced cell apoptosis, while remarkably inhibited cell migration, invasion as well as tumorigenicity in nude mice. Gene set enrichment analysis in Gene Expression Omnibus dataset revealed that apoptosis, epithelial-mesenchymal transition (EMT) and transforming growth factor-β (TGF-β) signaling pathway-related genes were enriched in TRIM66 higher expression patients, which was confirmed by western blot analysis in osteosarcoma cells with TRIM66 silenced. In conclusion, TRIM66 may act as an oncogene through suppressing apoptosis pathway and promoting TGF-β signaling in osteosarcoma carcinogenesis. TRIM66 may be a prognostic factor and potential therapeutic target in osteosarcoma.

Keywords: EMT; TGF-β; TRIM66; osteosarcoma; p53.

Figure 1. Correlation between TRIM66 expression and survival time of patients with osteosarcoma

A. TRIM66 mRNA level was significantly higher in osteosarcoma tissues (n = 45) than that in bone cysts (n = 14) from patients treated at Department of Orthopedic Surgery, Changzheng Hospital (Shanghai, China) between 2000 and 2006 (P < 0.0001); B. TRIM66 expression was significantly increased in osteosarcoma tumor tissues (n = 14) when compared with normal bone tissues (n = 4) of patients from GEO dataset E-MEXP-3628 (P < 0.05); C. The protein level of TRIM66 by immunohistochemistry in osteosarcoma tissues and bone cysts; D. The overall survival time of 101 patients with osteosarcoma; E. Survival analysis of patients from ArrayExpress dataset GSE 16102.

Figure 2. Depletion of TRIM66 inhibited cell growth in osteosarcoma cells

A. TRIM66 expression level in 5 osteosarcoma cell lines was analyzed by real-time PCR (right panel) and Western blot (left panel). Data were based on at least three independent experiments; B, C. Expression of TRIM66 in MG63 cells and HOS cells was analyzed by real-time PCR (middle panel) and Western blot (left panel). Cell proliferation (right panel) was detected 12, 24, 48 and 72 hours after transfection in MG63 and HOS cells. Data were based on at least three independent experiments, and shown as mean ± S.D. WT: wild type cells; NC: scrambled siRNA-transfected cells; RNAi: TRIM66-siRNA-transfected cells (***P < 0.001 as compared with NC).

Figure 3. Silencing of TRIM66 induced G0/G1 arrest and cell apoptosis in osteosarcoma cells

MG63 and HOS cells were transfected with indicated siRNA and 48 hours later cells were collected. A. Cell cycle profile was analyzed using flow cytometry; B. Cells were double stained with annexin V-FITC/PI and apoptosis rates was analyzed using flow cytometry. Data were based on at least three independent experiments, and shown as mean ± S.D. Representative images and quantitative results were shown. WT: wild type cells; NC: scrambled siRNA-transfected cells; RNAi: TRIM66-siRNA-transfected cells (**P < 0.01, ***P < 0.001 as compared with NC).

Figure 4. Silencing of TRIM66 inhibited cell migration and invasion in osteosarcoma cells

MG63 and HOS cells transfected with indicated siRNA and cell migration and invasion was analyzed as described in Materials and Methods A. knockdown of TRIM66 notably inhibited cell migration and invasion. Data were based on at least three independent experiments, and shown as mean ± S.D. Representative images and quantitative results were shown. WT: wild type cells; NC: scrambled siRNA-transfected cells; RNAi: TRIM66-siRNA-transfected cells (**P < 0.01, ***P < 0.001 as compared with NC).

Figure 5. Knockdown of TRIM66 in osteosarcoma cells reduced tumor growth in vivo

MG63 cells transfected with siRNA control (NC) or TRIM66-siRNA were subcutaneously injected in athymic nude mice. A. Tumor volume was evaluated for 45 days. Tumor growth was significantly slower in mice injected with TRIM66 knockdown cells. The mean value ± S.D. were from six animals in each group (**P < 0.001); B. Xenografts in nude mice at day 45; C. At day 45, mice were sacrificed and tumors were weighted. The six tumor tissues derived from the RNAi group were smaller than that from the control group; D. Western blot analysis of TRIM66, PCNA, Twist and MMP2 in xenografts from nude mice (**P < 0.01, ***P < 0.001 as compared with NC).

Figure 6. Apoptosis, EMT and TGF-β pathways were affected by TRIM66 siRNA treatment in osteosarcoma cells

A, B, C. GSEA was performed using E-MEXP-3628 dataset. Apoptosis, EMT and TGF-β pathway were identified with the strongest association with TRIM66-higher expression. Expression of three apoptosis pathway related gene was evaluated by Western blot in D. MG63 and E. HOS cells transfected with TRIM66-siRNA or siRNA control (NC). Expression of EMT pathway related gene was evaluated by Western blot in F. MG63 and G. HOS cells. Expression of TGF-β pathway related gene and phosphorylation of Smad2 and Smad4 was evaluated by Western blot in H, I. MG63 and J, K. HOS cells transfected with TRIM66-siRNA or siRNA control (NC). Data were based on at least three independent experiments, and shown as mean ± S.D. Representative images and quantitative results were shown. WT: wild type cells; NC: scrambled siRNA-transfected cells; RNAi: TRIM66-siRNA-transfected cells (*P < 0.05, **P < 0.01, ***P < 0.001 as compared with NC).