MicroRNA-302a inhibits osteosarcoma cell migration and invasion by directly targeting IGF-1R

Abstract

Osteosarcoma is one of the most frequent types of primary malignant bone neoplasm in children and adolescents. Despite advancements developed in therapeutic modalities, the 5-year overall survival rates for patients with metastatic osteosarcoma disease remain poor. The present study aimed to investigate the expression level of microRNA-302a (miR-302a) in osteosarcoma tissues and cell lines, and the biological roles of miR-302a in osteosarcoma cells. In addition, the molecular mechanism underlying its tumor suppressive roles was evaluated. miR-302a expression in osteosarcoma tissues and cell lines was detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Following transfection of miR-302a mimics or IGF-1R siRNA, transwell migration and invasion, luciferase reporter assay RT-qPCR and western blot assays were conducted in osteosarcoma cells. In the present study, the data demonstrated that miR-302a was frequently reduced in osteosarcoma tissue and cell lines. In addition, the expression of miR-302a was correlated with metastatic features of patients with osteosarcoma. Restoration of miR-302a expression significantly inhibited the migration and invasion capacity of osteosarcoma cells. Mechanistic studies indicated that insulin-like growth factor 1 receptor (IGF-1R) was a direct target gene of miR-302a. Overexpression of miR-302a resulted in decreased expression of IGF-1R at the mRNA and protein levels. Furthermore, the knockdown IGF-1R mimicked the functions of miR-302a overexpression on osteosarcoma cell migration and invasion. Collectively, the results of the current study indicate that miR-302a acts as a metastasis suppressing miRNA and could be investigated as a therapeutic target for the treatment of patients with osteosarcoma to prevent metastasis.

Keywords: insulin-like growth factor 1 receptor; invasion; metastasis; microRNA-302a; migration; osteosarcoma.

Figure 1.

Expression levels of miR-302a in OS. (A) The expression of miR-302a in OS tissues was significantly decreased compared with matched NATs. (B) The levels of miR-302a in OS tissue samples with metastatic disease were significantly downregulated compared with that in OS tissues without metastasis. (C) miR-302a expression levels in OS cell lines (HOS, MG63, SAOS2 and U2OS) were lower compared with that in the human normal osteoblastic hFOB 1.19 cell line. *P<0.05 compared with their respective controls. Data are presented as the mean ± standard deviation. miR, microRNA; OS, osteosarcoma; NATs, matched normal adjacent tissues.

Figure 2.

Overexpression of miR-302a suppressed the migration and invasion abilities of osteosarcoma cell lines in vitro. (A) miR-302a was significantly upregulated in MG63 and U2OS cells following the transfection with miR-302a mimics. (B) Representative image of the migration and invasion assays performed on MG63 and U2OS cells. (C) Transfection of miR-302a mimics significantly suppressed MG63 and U2OS cells migration and invasion capacity compared with cells transfected with the NC. *P<0.05 compared with their respective controls. Data are presented as the mean ± standard deviation. miR, microRNA; NC, negative control.

Figure 3.

IGF-1R was predicted as a target of miR-302a in osteosarcoma. (A) Computational analysis using miRanda and TargetScan revealed that miR-302a potentially targeted IGF-1R. (B) MG63 and U2OS cells were transfected with miR-302a mimics or NC, and IGF-1R 3′ UTR Wt or Mut luciferase reporter vector. (C) Expression levels of IGF-1R mRNA were measured using reverse transcription-quantitative polymerase chain reaction in MG63 and U2OS cells transfected with miR-302a mimics or NC. Transfection with miR-203a mimic resulted in a significant downregulation of IFG-1R mRNA compared with the negative control. (D) Protein levels of IGF-1R were detected using western blot analysis in MG63 and U2OS cells transfected with miR-302a mimics or NC. Transfection with miR-203a mimic resulted in a marked downregulation of IFG-1R protein expression compared with the negative control. *P<0.05 compared with their respective controls. Data are presented as the mean ± standard deviation. miR, microRNA; NC, negative control; IGF-1R, insulin-like growth factor 1 receptor; UTR, untranslated region; wt, wildtype; mut, mutant.

Figure 4.

Knockdown IGF-1R mimicked functions with miR-302a overexpression of OS cells. (A) Protein levels of IGF-1R were detected using western blot analysis in MG63 and U2OS cells transfected with IGF-1R or control siRNA. (B) Representative image of the Transwell migration and invasion assays for OS cells treated with IGF-1R or control siRNA. (C) Transfection of IGF-1R siRNA significantly inhibited MG63 and U2OS cell migration, and invasion capacity compared with control treated cells. *P<0.05 compared with their respective controls. Data are presented as the mean ± standard deviation. miR, microRNA; siRNA, small interfering RNA; IGF-1R, insulin-like growth factor 1 receptor; OS, osteosarcoma.