miR-129通过靶向SEPHS1对骨肉瘤细胞MG-63增殖及凋亡的影响

Effect of miR-129 on proliferation and apoptosis of osteosarcoma MG-63 cells by targeting SEPHS1

目的探讨miR-129对骨肉瘤细胞系MG-63细胞活性及凋亡的影响。方法收集30例骨肉瘤组织及其配对的癌旁组织,应用实时荧光定量法(RT-PCR)检测miR-129 miRNA和SEPHS1的mRNA的表达水平,应用蛋白质免疫印迹法(Western blot)检测SEPHS1蛋白的表达水平。在体外培养的MG-63细胞中过表达或敲低miR-129,通过CCK-8检测细胞增殖情况,检测miR-129对MG-63细胞增殖能力的影响。同时应用Western blot和Hoechest染色检测MG-63的凋亡情况。结果在本研究中,我们发现miR-129在骨肉瘤组织中的表达显著高于癌旁组织(P <0. 05)。通过建立miR-129过表达或敲低的体外模型,确定miR-129模拟物和抑制物浓度为50 n M和200 n M时过表达和敲低的效果最佳。通过软件预测miR-129和SEPHS1具有结合的可能,荧光素酶报告基因试验进一步确定了miR-129和SEPHS1的结合关系。通过Western blot和Hoechest试验,发现敲低miR-129可以显著抑制MG-63的活性,加速MG-63的凋亡。结论 miR-129可能通过与SEPHS1结合,在调节骨肉瘤细胞增殖和凋亡进程中起关键作用。

Objective The aim of this study was to investigate the effect of miR-129 on the proliferative activity and apoptosis of osteosarcoma MG-63 cells.Methods Thirty cases of osteosarcoma and its adjacent paracancerous tissues were collected.RT-PCR was used to detect the expressions of miR-129 mRNA and SEPHS1 mRNA.Western blot was used to detect the protein level of SEPHS1.After MiR-129 was over-expressed and knocked down,the cell proliferation was detected in MG-63 cells by CCK-8,and apoptosis was detected in MG-63 cells by Westerm blot and hoechest staining.Results In this study,the expression of miR-129 in osteosarcoma tissues was significantly higher than that in para-cancerous tissues(P<0.05).Through the established model in vitro of miR-129 overexpression or knockdown,it was determined that the miR-129 mimetic and inhibitor concentrations were optimal for overexpression and knockdown at 50 nM and 200 nM,respectively.The possibility of binding between miR-129 and SEPHS1 was predicted by software,and the luciferase reporter assay further confirmed the binding relationship between miR-129 and SEPHS1.Knockdown of miR-129 significantly inhibited the proliferation of MG-63 cells and accelerated the apoptosis of MG-63 cells.Conclusion miR-129 plays a key role in regulating the proliferation and apoptosis of osteosarcoma cells by binding to SEPHS1.