慢病毒介导miR-145对骨肉瘤MG-63细胞侵袭和迁移力的影响

Effects of Lentivirus-mediated miR-145 on Invasion and Migration of Osteosarcoma MG-63 Cells

目的探讨慢病毒介导miR-145对成骨肉瘤细胞MG-63侵袭及迁移能力的影响。方法通过慢病毒携带miR-145表达载体转染骨肉瘤MG-63细胞,实验分为:转染组(转染miR-145阳性序列)、对照组(转染阴性对照序列)、空白组(PBS)。利用生物信息学软件预测血管内皮生长因子(VEGF)为miR-145的候选靶基因,利用双荧光素酶报告基因检测验证miR-145对靶基因VEGF的直接调控作用。实时荧光定量PCR检测基因表达,Western-blot检测蛋白表达,Transwell小室侵袭实验检测细胞侵袭能力,细胞划痕实验检测细胞迁移运动能力。结果与对照组比较,转染组细胞内荧光素酶活性明显下降(P<0.05);与对照组和空白组比较,转染组miR-145mRNA的表达量显著增加(P<0.05),VEGF mRNA及蛋白表达量显著减少(P<0.05),穿膜细胞数及细胞划痕愈合率均显著降低(P<0.05)。结论 miR-145通过靶向VEGF基因,进而抑制骨肉瘤MG-63细胞的侵袭及迁移能力。

Objective To investigate the effect of lentivirus-mediated miR-145 on the invasion and migration of osteosarcoma MG-63 cells. Methods Lentiviral-mediated miR-145 expression vector was transfected into osteosarcoma MG-63 cells. The experiment was divided into transfection group(transfected miRNA-145 positive sequence),nonsense sequence group(transfected negative control sequence),and control group(blank PBS). Bioinformatics software was used to predict VEGF as a candidate target gene for miR-145,Dual-Luciferase reporter assay to verify the direct regulation of miR-145 on target gene VEGF. The mRNA expression of miR-145 and VEGF was detected by real-time fluorescence quantitative PCR. The expression of VEGF protein was detected by Western-blot,cell invasion was detected by Transwell chamber invasion assay,while cell migration ability was detected by cell scratches test. Results Compared with the control group,the luciferase activity in transfection group was significantly decreased(P<0.05);compared with the control group and the blank group,the expression of miR-145 mRNA was significantly increased in the transfection group(P<0.05),while the expression of VEGF mRNA was significantly decreased(P<0.05). Compared with the control group and the blank group,the expression of VEGF protein was significantly decreased in the transfection group(P<0.05). Compared with the control group and blank group,the number of transmembrane cells in transfection group was significantly decreased(P<0.05). Compared with the control group and blank group,the healing rate of the cells in transfection group was significantly decreased(P<0.05). Conclusion miR-145,through the action of the VEGF target gene,inhibit the invasion and migration of osteosarcoma MG-63 cells.