The effect of GAS5 on proliferation, migration and invasion of osteosarcoma U2OS cells

English Keywords:Osteosarcoma  Growth arrest-specific 5  MicroRNA-221  Matrix metalloproteinase inhibitor-2  Proliferation  Migration  Invasion

English Abstract:

  【Abstract】 Objectives: To observe the effect of growth arrest-specific 5(GAS5) on the proliferation, migration and invasion of osteosarcoma U2OS cells and its mechanism. Methods: In human osteosarcoma U2OS cells and normal human osteoblast hFOB 1.19 cells, to detect the expressions of GAS5 and miR-221 by qRT-PCR, and tissue inhibitor of metalloproteinase 2(TIMP2) protein by Western blot. After transfection of pcDNA-GAS5 recombinant plasmid, the effects of GAS5 on the proliferation, migration and invasion of U2OS cells were detected by MTT assay and Transwell chamber assay, and the effect on the expression of miR-221 was analyzed by qRT-PCR. The targeting relationship between GAS5 and miR-221, as well as between miR-221 and TIMP2 was predicted by Starbase software and validated by double luciferase reporter gene experiments. After transfection of miR-221 mimics and miR-221 inhibitor, the effects of miR-221 over-expression on the proliferation, migration and invasion of U2OS cells regulated by GAS5 and effect of miR-221 on the expression of TIMP2 protein were observed. Results: Compared with hFOB 1.19, the expression levels of GAS5 and TIMP2 in U2OS cells were decreased significantly, while the expression level of miR-221 was increased significantly (GAS5: P=0.0001; TIMP2: P=0.0003; miR-221: P=0.0004). The GAS5 over-expression inhibited the proliferation, migration and invasion of U2OS cells (proliferation 48h: P=0.0005; 72h: P=0.0002; migration: P=0.002; invasion: P=0.001), while the miR-221 over-expression significantly reversed the inhibitory effect of GAS5 on the proliferation, migration and invasion of U2OS cells (proliferation 48h: P=0.0002; 72h: P=0.0003; migration: P=0.0001; invasion: P=0.0001). Starbase predicted that there was a complementary binding site between miR-221 and GAS5, and there were also sites that could complement TIMP2 3′UTR. Double luciferase results showed that the luciferase activity of wild GAS5 (GAS5-WT) and TIMP2 (TIMP2-WT) cells significantly decreased after over-expression of miR-221 (P both=0.0003). At the same time, under the condition of GAS5 over-expression, the expression level of miR-221 in U2OS cells decreased significantly (P=0.0001), whereas it increased significantly (P=0.0001) under contrary condition. Similarly, with miR-221 over-expression, the expression level of TIMP2 protein decreased significantly(P=0.0003), and conversely, it increased significantly(P=0.0009). Conclusions: GAS5 promotes TIMP2 expression by targeting inhibition of miR-221, thereby inhibiting the proliferation, migration and invasion of U2OS cells