GAS5对骨肉瘤U2OS细胞增殖、迁移和侵袭的影响

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

目的:观察生长停滞特异性转录因子5(GAS5)对骨肉瘤U2OS细胞增殖、迁移和侵袭的影响,探讨其机制。方法:在人骨肉瘤U2OS细胞和人正常成骨hFOB 1.19细胞中,采用实时定量PCR(qRT-PCR)检测GAS5和微小RNA (miR)-221的表达,Western blot检测基质金属蛋白酶抑制物-2 (TIMP2)蛋白的表达。转染pcDNA-GAS5重组质粒后,采用噻唑蓝(MTT)法和Trasnwell小室法检测GAS5对U2OS细胞增殖、迁移和侵袭的影响,qRT-PCR检测GAS5对miR-221表达的影响。利用Starbase软件预测和双荧光素酶报告基因实验验证GAS5与miR-221以及miR-221与TIMP2的靶向关系。转染miR-221模拟物和miR-221抑制剂后,观察miR-221过表达对GAS5调控的U2OS细胞增殖、迁移和侵袭的影响以及miR-221对TIMP2蛋白表达的影响。结果:与正常hFOB 1.19细胞相比,U2OS细胞中GAS5和TIMP2蛋白的表达水平明显降低,而miR-221表达水平显著升高(GAS5:P=0.0001;TIMP2:P=0.0003;miR-221:P=0.0004)。GAS5过表达可抑制U2OS细胞增殖、迁移和侵袭(增殖48h:P=0.0005;增殖72h:P=0.0002;迁移:P=0.002;侵袭:P=0.001),而miR-221过表达可明显逆转GAS5对U2OS细胞增殖、迁移和侵袭的抑制作用(增殖48h:P=0.0002;增殖72h:P=0.0003;迁移:P=0.0001;侵袭:P=0.0001)。Starbase软件预测到miR-221存在能够与GAS5互补结合的位点,还存在能够与TIMP2 3′UTR互补结合的位点;双荧光素酶结果显示miR-221过表达后野生型GAS5(GAS5-WT)和野生型TIMP2 (TIMP2-WT)细胞的荧光素酶活性明显降低(P均为0.0003);同时,GAS5过表达后,U2OS细胞中miR-221表达水平明显降低(P=0.0001),反之miR-221明显升高(P=0.0001);miR-221过表达后,U2OS细胞中TIMP2蛋白的表达水平明显降低(P=0.0003),反之TIMP2蛋白的表达水平明显升高(P=0.0009)。结论:GAS5可通过靶向抑制miR-221促进TIMP2表达,进而抑制U2OS细胞的增殖、迁移和侵袭。

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.