MiR-1 Suppresses Proliferation of Osteosarcoma Cells by Up-regulating p21 via PAX3

Abstract

Background/aim: miRNA-1(miR-1) is down-regulated in various cancer cells including osteosarcoma cells. This study was conducted to analyze the function of miR-1 in osteosarcoma cells.

Materials and methods: miR-1 expression in osteosarcoma cells was evaluated by qRT-PCR. Cell proliferation was evaluated after transfecting miR-1 by WST8 assay and FACS analysis, both in vitro and in vivo.

Results: Overexpression of miR-1 suppressed cell proliferation and induced cell-cycle arrest in the G₀-G₁ phase by increasing p21 levels via a p53-independent pathway. Overexpression of miR-1 down-regulated PAX3, a potential p21-regulating gene. Moreover, knockdown of PAX3 suppressed cell proliferation by increasing p21 levels, and induced arrest at the G₀/G₁ phase. Administration of miR-1 showed an in vivo antitumor effect.

Conclusion: Overexpression of miR-1 suppressed cell proliferation and induced arrest in the G₀/G₁ phase by increasing p21 levels via a p53-independent pathway through PAX3 suppression. These results indicate that miR-1 could be a therapeutic target for osteosarcoma.

Keywords: Osteosarcoma; PAX3; miR-1; p21; p53-independent pathway.

Figure 1. Expression level of miR-1. A: Expression levels of miR-1 in the osteosarcoma cell lines (MG63, Saos2, G292) and normal cell line (hFOB1.19). B: Changes in miR-1 expression levels 48 h after transfection of the osteosarcoma cell line MG63 with miR-1 (50 nM). *p<0.05.

Figure 2. Evaluation of cell proliferation and cell cycle after transfection of miR-1 and NC-miRNA in the osteosarcoma cell line. A: Cell proliferation at 24, 48, and 72 hs. B: Phase of cell cycle at 48 h. C: Analysis of p21 expression levels by qRT-PCR. D: Western blot analysis of the expression of p21, p53, phosphorylated p53, and p73 at 48 h by. *p<0.05, **p<0.01.

Figure 3. The effect of transfection of miR-1 or NC-miRNA in the osteosarcoma cell line (MG63) on PAX3 levels. A: The miR-1 sequence and target area in PAX3. B: qRT-PCR analysis of PAX3 expression at 48 h and C: western blot analysis of PAX3 expression at 48 h.

Figure 4. Evaluation of cell proliferation and cell cycle after transfection of siPAX3 and NC-siRNA (10 nM) in the osteosarcoma cell line. A: qRTPCR analyses of PAX3 expression at 48 h. B: Western blot analysis of PAX3 expression at 48 h. C: Cell proliferation at 24, 48, and 72 h. D: Distribution of cells in each phase of the cell cycle at 48 h. E: qRT-PCR analysis of p21 expression levels and F: western blot analysis of p21 expression levels. *p<0.05, **p<0.01.

Figure 5. The mouse subcutaneous tumor model was prepared, and miR-1 or NC-miRNA were administered around the tumor (each n=6). miRNA 3 times on Days 0, 7, and 14. A: Changes in tumor volume over time. B: Tumor appearance C: Ki67 staining of tumor tissues. D: Evaluation of proliferation activity by scoring. *p<0.05, **p<0.01.