miR-499-5p调控AKT2基因对骨肉瘤MG63细胞凋亡和迁移的影响

Role of miR-499-5p in osteosarcoma MG63 cells apoptosis and migration by regulating AKT2 expression

关键词:  微小RNA  丝氨酸/苏氨酸蛋白激酶2  骨肉瘤MG63细胞

Key words:  miRNA  AKT2  osteosarcoma MG63 cells

摘要:

目的　探讨miR-499-5p在骨肉瘤MG63细胞中的表达及其对MG63细胞凋亡和迁移的影响。　方法　运用生物信息学软件TargetScan和miRanda对miR-499-5p和AKT2基因的靶向配对关系进行预测。未转染细胞设为空白对照组（control组），脂质体2000转染miR-499-5p模拟物为mimics组，转染干扰AKT2基因的siRNA为siRNA组。 Real-time PCR检测3组细胞中miR-499-5p和AKT2 mRNA的表达情况，western blot检测AKT2蛋白的表达情况，流式细胞术检测3组细胞凋亡情况，细胞划痕实验检测体外的迁移情况。　结果　TargetScan和miRanda显示miR-499-5p和AKT2基因二者靶向配对良好。Real-time PCR检测结果表明转染mimics后，AKT2 mRNA的表达量降至（45.06±8.12）%，与control组（100%）比较，差异有显著性意义（P<0.05）。Western blot检测结果表明mimics组AKT2蛋白相对表达量为（0.32±0.05)，与comtrol组（0.69±0.11）比较，明显降低，P<0.05。流式细胞术和细胞划痕实验结果显示mimics组MG63细胞凋亡率（18.97±2.41）%及细胞划痕愈合率（63.54±5.43）%，与control组（10.35±2.56）%和（90.35±6.81）%相比差异均有显著性意义（P＜0.05）。　结论　miR-499-5p能够下调骨肉瘤MG63细胞中AKT2基因的表达，促进癌细胞的凋亡以及抑制细胞的迁移。

Abstract:

Objective　 To identify expressions of miR-499-5p and AKT2 in osteosarcoma MG63 cells and explore the role of miR-499-5p on cell apoptosis and migration which may regulate AKT2 expression.　Methods　 It was predicted by bioinformatics that miR-499-5p may regulate AKT2 expression. After transfection of miR-499-5p mimics or siRNA into cells, the expressions of miR-499-5p and AKT2 were determined by real-time PCR and western blot. The apoptosis and migration of MG63 cells were detected in vitro by flow cytometry and the wound healing assay respectively.　Results　 MiRanda and TargetScan showed that miR-499-5p was well complementary with AKT2 gene. Compared with the control group, the expressions of AKT2 mRNA （45.06±8.12）% and protein（0.32±0.05）decreased when miR-499-5p over-expression（P＜0.05）. Compared with the control group, the apoptosis（18.97±2.41）% of MG63 cells was promoted and the migration（63.54±5.43）% was suppressed after transfection of miR-499-5p（P＜0.05）.　Conclusion　 MiR-499-5p may down- regulate AKT2 expression in osteosarcoma MG63 cells, which could play a role in promoting cell apoptosis and inhibiting cell migration.