miR-17-92基因簇对骨肉瘤细胞增殖侵袭的影响

Effects of miR-17-92 gene cluster on proliferation and invasion of osteosarcoma cells

摘要:

[目的] 探究 miR-17-92 基因簇对骨肉瘤 （osteosarcoma, OS） 细胞增殖、侵袭的影响及可能机制。[方法] qRTPCR 检测 OS 细胞 MG-63 转染前 （MG-63 细胞）、人成骨细胞 hFOB1.19 （hFOB 细胞） 中的 miR-17-92 基因簇水平。分别对 MG-63 细胞行 miR-17-92 基因簇模拟物组转染（miR-17-92 组）与阴性对照转染（miR-NC 组），比较两组 miR-17-92 基因簇水平、细胞增殖水平及侵袭能力，及转染后 MG-63 细胞转化生长因子-β1（transforming growth factor beta 1, TGF-β1）、Smad3、 基质金属蛋白酶 （matrix metalloproteinase, MMP） -2、MMP-9 蛋白水平。[结果]MG-63 细胞的 miR-17 [(4.1±1.0) vs (1.2±0.3), P＜0.05]、miR-18a [(2.3±0.5) vs (1.4±0.5), P＜0.05]、miR-19a [(2.0±0.4) vs (1.3±0.4), P＜0.05]、miR-19b [(2.1±0.4) vs (1.2±0.3), P＜ 0.05]、miR-20 [(6.2±2.2) vs (1.4±0.4), P＜0.05] 和 miR-92 [(5.1±1.9) vs (1.7±0.5), P＜0.05] 相对表达量均显著高于 hFOB 细胞。miR17-92 组的 miR-17 [(7.1±2.2) vs 4.1±1.3), P＜0.05]、miR-18a [(3.6±0.6) vs (2.3±0.7), P＜0.05]、miR-19a [(4.4±1.0) vs (2.1±0.5), P＜ 0.05]、miR-19b [(4.6±1.0) vs (2.2±0.5), P＜0.05]、miR-20 [(9.3±2.1) vs (6.2±2.0), P＜0.05] 和 miR-92 [(8.7±2.3) vs (5.0±1.4), P＜0.05] 相对表达量均显著高于 miR-NC 组；miR-17-92 组 OD450 值及侵袭细胞数均显著多于 miR-NC 组（P＜0.05）；miR-17-92 组 TGF-β 1、Smad3、MMP-2、MMP-9 蛋白表达水平均显著高于 miR-NC 组（P＜0.05）。[结论]骨肉瘤 MG-63 细胞中 miR-17-92 基因簇异常高表达，可增强细胞增殖及侵袭能力，其机制可能与其激活 TGF-β1/Smad3/MMP-9 信号通路有关。

Abstract:

[Objective] To explore the effects of miR-17-92 gene cluster on proliferation and invasion of osteosarcoma (OS) cells and its possible mechanism. [Methods] The level of miR-17-92 gene cluster in OS MG-63 cells before transfection and human osteoblasts hFOB1.19 (hFOB cells) was detected by qRT- PCR. MG- 63 cells were transfected with miR- 17-92 gene cluster mimics (miR- 17-92 group) and negative control transfection (miR-NC group), respectively. The levels of miR-17-92 gene cluster, cell proliferation and invasion ability of the two groups were compared. In addition, the transforming growth factor beta 1 (TGF-β1), Smad3, matrix metalloproteinase (MMP) -2 and MMP-9 protein levels were also measured and compared. [Results] The MG-63 cells had significantly higher relative ex- pression level of miR-17 [(4.1±1.0) vs (1.2± 0.3), P＜0.05], miR-18a [(2.3±0.5) vs (1.4±0.5), P＜0.05], miR -19a [(2.0±0.4) vs (1.3±0.4), P＜ 0.05], miR-19b [(2.1±0.4) vs (1.2±0.3), P＜0.05], miR-20 [(6.2±2.2) vs (1.4±0.4), P＜0.05] and miR-92 [(5.1±1.9) vs (1.7±0.5), P＜0.05] than the hFOB cells. After transfection, the MiR-17-92 group had significantly higher relative expression level of miR-17 [(7.1±2.2) vs (4.1± 1.3), P＜0.05], miR-18a [(3.6±0.6) vs (2.3± 0.7), P＜0.05], miR-19 a [(4.4±1.0) vs (2.1±0.5), P＜0.05], miR-19 b [(4.6±1.0) vs (2.2±0.5), P＜ 0.05], miR-20 [(9.3±2.1) vs 6.2±2.0), P＜0.05] and miR-92 [(8.7±2.3) vs (5.0±1.4), P＜0.05] than the miR-NC group. In addition, the miR17-92 group had significantly higher OD450 value and the number of invasive cells than the miR-NC group (P＜0.05). Furthermore, the the miR-17-92 group had significantly higher protein expression levels of TGF-β1, Smad3, MMP-2 and MMP-9 than the miR-NC group (P＜ 0.05). [Conclusion] The abnormally high expression of miR-17-92 gene cluster in osteosarcoma MG-63 cells can enhance cell prolifera- tion and invasion, and its mechanism may be related to the activation of TGF-β1/Smad3/MMP-9 signaling pathway.