CTGF increases matrix metalloproteinases expression and subsequently promotes tumor metastasis in human osteosarcoma through down-regulating miR-519d

Abstract

Osteosarcoma, the most common primary malignant bone tumor, shows potent capacity for local invasion and distant metastasis. Connective tissue growth factor (CTGF/CCN2), a secreted protein, binds to integrins, modulates invasive behavior of certain human cancer cells. Effect of CTGF in metastasis of human osteosarcoma is unknown. We found overexpression of CTGF increasing matrix metalloproteinases (MMPs)-2 and MMP-3 expression as well as promoting cell migration. MicroRNA (miRNA) analysis of CTGF-overexpressed osteosarcoma versus control cells probed mechanisms of CTGF-mediated promotion of migration. Among miRNAs regulated by CTGF, miR-519d was most downregulated after CTGF treatment. Co-transfection with miR-519d mimic reversed CTGF-mediated MMPs expression and cell migration. Also, MEK and ERK inhibitors or mutants reduced CTGF-increased cell migration and miR-519d suppression. By contrast, knockdown of CTGF diminished lung metastasis in vivo. Clinical samples indicate CTGF expression as linked with clinical stage and tumor metastasis. Taken together, data show CTGF elevating MMPs expression and subsequently promoting tumor metastasis in human osteosarcoma, down-regulating miR-519d via MEK and ERK pathways, making CTGF a new molecular therapeutic target in osteosarcoma metastasis.

Figure 1. CTGF promotes migration in human osteosarcoma cells

(A&B) CTGF expression and migratory activity in indicated cells was examined by western blot and Transwell assay. (C&E) CTGF-overexpressing MG-63 cells were established, using pCDNA3.1 vector. Protein and mRNA expression of CTGF and migratory activity were examined by western blot, q-PCR and Transwell assay. (D) Protein and mRNA expression of CTGF in indicated cells were examined by western blot and q-PCR. (F) U-2 OS/CTGF shRNA cells were incubated with vehicle or recombine protein of CTGF (rCTGF; 50 ng/ml), cell migration examined by Transwell assay, results expressed as mean ± SEM. *, p < 0.05 as compared with controls, and ^#, p < 0.05 as compared with U-2 OS/CTGF shRNA group.

Figure 2. CTGF increases cell migration by suppressing miR-519d expression

(A) The miR-519d expression in indicated cells was examined by qRT-PCR. (B) MG-63 cells and U-2 OS cells were incubated with rCTGF (5-50 ng/ml) for 24 h, miR-519d expression examined by qRT-PCR. (C) Cells were transfected with miR-519d mimic or inhibitor for 24 h and cell migration examined by Transwell assay. Results are expressed as mean ± SEM. *, p < 0.05 as compared with controls, and ^#, p < 0.05 as compared with CTGF-treated control group.

Figure 3. CTGF increases MMP-2 and MMP-3 expression and cell migration by down-regulating miR-519d

(A) Cells were transfected with MMP-2 or MMP-3 siRNA for 24 h. The MMP2 and MMP-3 expression and cell migration were examined by western blot and Transwell assay. (B-D) The mRNA and protein expression in indicated cells was examined by q-PCR and western blot. (E) Cells were transfected with miR-519d mimic or inhibitor for 24 h and MMPs expression was examined by western blot and q-PCR. (F) Cells were transfected with indicated luciferase plasmid for 24 h, and the relative luciferase activity was measured. Results are expressed as mean ± SEM. *, p < 0.05 as compared with control group, and ^#, p < 0.05 as compared with CTGF-treated control group.

Figure 4. MEK/ERK pathway is involved in CTGF-induce migration and suppression of miRNA-519d

(A-D) Cells were pretreated with PD98059 (10 μM) and U0126 (10 μM) for 30 min or transfected with MEK1 and ERK2 mutant for 24 h, the MMPs expression and cell migration was examined by q-PCR, ELISA, and Transwell assay. (E) MG-63 cells were incubated with rCTGF (50 ng/ml) for indicated time intervals, MEK and ERK phosphorylation examined by western blot. (F) Cells were pretreated with PD98059 (10 μM) and U0126 (10 μM) for 30 min or transfected with MEK1 and ERK2 mutant for 24 h followed, the miR-519d expression was examined by qRT-PCR. Results expressed as mean ± SEM. *, p < 0.05 as compared with controls, and ^#, p < 0.05 as compared with CTGF-treated control gruop.

Figure 5. Knockdown of CTGF expression suppress lung metastasis in vivo

(A) U-2 OS/Luc or U-2 OS/shCTGF-Luc cells were injected into the lateral tail vein, and lung metastasis was monitored by bioluminescence imaging at indicated time intervals. (B-E) After 6 weeks mice were sacrificed, their lungs were excised and photographed, tumor colonies counted and stained with CTGF, MMP-2, and MMP-3. Results are expressed as mean ± SEM. *, p < 0.05 as compared with U-2 OS/Luc group.

Figure 6. Clinical importance of CTGF, MMPs and miR-519d in osteosarcoma

(A) IHC stain of CTGF, MMP-2, and MMP-3 in normal versus osteosarcoma tissue. Correlation between CTGF/MMP-2 (B), CTGF/MMP-3 (C) and CTGF/mi-519d (D) in human osteosarcoma.