MiRNA-15a inhibits proliferation, migration and invasion by targeting TNFAIP1 in human osteosarcoma cells

Abstract

Recent data strongly suggest the important role of miRNAs in various cancer-related processes. Osteosarcoma is the most common type of primary malignant bone tumor and is characterized by complex genetic changes and resistance to conventional treatments. In this study, the role of miRNA-15a (miR-15a) in the progression and metastasis of osteosarcoma was investigated. The result demonstrated that the expression of miR-15a was down-regulated in osteosarcoma tissues and cell lines as compared with that in adjacent non-neoplastic bone tissues and the osteoblastic cell line. In functional assays, miR-15a inhibited cell proliferation, migration and invasion in U2OS and MG-63 cells. Meanwhile, bioinformatic analysis combined with experimental confirmation demonstrated that tumor necrosis factor; α-induced protein 1 (TNFAIP1) gene is a potential target of miR-15a and can be directly regulated by miR-15a. Down-regulation of TNFAIP1 induced effects on osteosarcoma cell lines similar to those induced by miR-15a. Taken together, these data suggest that miR-15a may act as a tumor suppressor, which is commonly down-regulated in both osteosarcoma tissues and cells. TNFAIP1 plays an important role in mediating miR-15a dependent biological functions in osteosarcoma. Reintroduction of miR-15a may be a novel therapeutic strategy by down-regulating TNFAIP1 expression.

Keywords: invasion; miRNA-15a (miR-15a); migration; osteosarcoma; proliferation; tumor necrosis factor; α-induced protein 1 (TNFAIP1).

Figure 1

miR-15a is down-regulated in osteosarcoma. A. The relative expression levels were determined by qRT-PCR in human osteoblast cell line (hFOB) and osteosarcoma cells (MG-63 and U2OS). *P < 0.05 vs. hFOB. B. qRT-PCR analysis of miR-15a expression in 45 pairs osteosarcoma tissues and their corresponding adjacent normal bone tissues. The expression of miR-15a was normalized to U6. *P < 0.05 vs. normal tissues.

Figure 2

miR-15a inhibits osteosarcoma cell proliferation. (A) qRT-PCR analysis of miR-15a expression after the transfection of miR-15a mimics or scramble or no transfection. (B) qRT-PCR analysis of miR-15a expression after the transfection of miR-15a inhibitors or scramble or no transfection. Proliferation curves of MG-63 (C) and U2OS (D) cells, respectively. The effect of miR-15a on cell proliferation was evaluated by CCK-8 assay after miR-15a transfection of MG-63 and U2OS. *P < 0.05 vs. untreated or scramble.

Figure 3

miR-15a inhibits osteosarcoma cell migration and invasion. A. Migration assays of the MG-63 and U2OS cells after treatment with miR-15a mimics, inhibitors or scramble or no transfection. B. Invasion analysis of the MG-63 and U2OS cells after treatment with miR-15a mimics, inhibitors or scramble or no transfection. Each bar represents the mean ± SD. The results were reproduced in three independent experiments. *P < 0.05 vs. untreated or scramble, #P < 0.05 vs. untreated or scramble.

Figure 4

miR-15a directly targets TNFAIP1 by binding to its 3’-UTR. A. The predicted miR-15a binding site within the TNFAIP1 3’-UTR and a mutated version generated by site directed mutagenesis are shown. B. Luciferase reporter assay illustrating direct binding of miR-15a to the WT, but not Mut sequences within the 3’-UTR of TNFAIP1. C. mRNA levels of TNFAIP1 were determined by real-time PCR in transfected MG-63 cells. D. Western blots were used to confirm the expression of TNFAIP1 in MG-63 cells after transfection; GAPDH was used as a control. The statistical analysis was based on three independent experiments. Error bars represent mean ± SD. *P < 0.05 vs. untreated or scramble, #P < 0.05 vs. untreated or scramble.

Figure 5

TNFAIP1 is involved in miR-15a-inhibited growth, migration and invasion in MG-63 cells. A. qRT-PCR and western blotting analysis of TNFAIP1 expression in MG-63 cells transfected with TNFAIP1 siRNA or negative control (NC). B and C. Down-regulation of TNFAIP1 suppressed cell proliferation, migration and invasion of MG-63 cells. The values represent the mean ± SD of three replicates. *P < 0.05 vs. NC.