微小RNA-499-5p对骨肉瘤细胞增殖能力的影响

The effect of microRNA - 499 - 5p on osteosarcoma cell growth in vitro

目的检测微小RNA-499-5p（miR-499-5p）在骨肉瘤组织中的表达及其对骨肉瘤细胞增殖能力的影响，并探讨其调节机制。方法实时定量聚合酶链反应（PCR）法用来检测骨肉瘤组织、瘤旁组织及骨肉瘤细胞株中miR-499-5p的表达量。细胞计数试剂盒（CCK-8）法及Western blot分别用来研究miR-499-5p对骨肉瘤细胞增殖能力的影响及探讨miR-499-5p调节骨肉瘤细胞增殖的相关机制。结果在骨肉瘤组织中，miR-499-5p表达量显著升高（肉瘤旁组织中表达量为0.34±0.09，而骨肉瘤组织中的表达量为1.47±0.18，P＝0.008）；此外，miR-499-5p在骨肉瘤细胞株HoS、MG-63及Hu09中的水平（分别为1.65±0.03、1.13±0.11、1.87±0.14）显著高于其在人骨髓间充质干细胞hMSCs中的水平（0.56±0.19，P＝0.005）；CCK-8实验结果表明，与空白及阴性对照组比较，在转染miR-499-5p mimics后48、72、96 h，MG-63细胞的增殖能力明显增强（P＝0.030）；流式细胞术检测细胞凋亡的结果表明，过表达miR-499-5p组内MG-63细胞的凋亡率为（0.87±0.19）%，显著低于空白组及阴性对照组内细胞的凋亡率[分别为（5.49±0.53）%、（6.72±0.85）%，P＝0.030]；此外，我们通过靶基因预测软件、结合RT-qPCT及Western blot结果初步认为miR-499-5p可通过转录后调节程序性细胞死亡因子4（PDCD4）的表达而发挥上述促癌效应。结论miR-499-5p在骨肉瘤组织中高表达且其在体外可促进骨肉瘤细胞的增殖。

ObjectiveTo explore microRNA-499-5p （miR-499-5p） expression and its function in osteosarcoma tissues and cells. MethodsmiR-499-5p levels were examined with real-time quantitative reverse transcriptase-polymerase chain reaction （RT-qPCR） in 20 paired osteosarcoma samples, and three osteosarcoma cell lines. Then the effects of miR-499-5p on osteosarcoma cell proliferation and apoptosis were detected by cell counting kit-8 （CCK-8） Kit and flow cytometry assays. Finally, RT-qPCR and Western blotting were applied to investigate the target gene of miR-499-5p in osteosarcoma cells. ResultsmiR-499-5p expression was significantly upregulated in osteosarcoma tissues when compared with their matched normal parts（1.47±0.18 vs. 0.34±0.09, P=0.008）; Consistent with above results, overexpressed miR-499-5p levels were also detectd in three osteosarcoma cell lines （HoS, MG-63, Hu09） when compared with human mesenchymal stem cells （hMSCs） （1.65±0.03, 1.13±0.11, 1.87±0.14 vs. 0.56±0.19, respectively, P=0.005）. In addition, miR-499-5p may act an oncogene by negatively regulating programmed cell death 4 （PDCD4） expression： Overexpressed miR-499-5p obviously prompted MG-63 cell proliferation rate （P=0.030）; Moreover, 48 h after transfection, MG-63 cells apoptosis rate in miR-499-5p-treat group was （0.87±0.19）%, significantly reduced when compared with blank group and miR-control group [（5.49±0.53）%, （6.72±0.85）%, respectively, P=0.030].ConclusionOur data reveal that miR-499-5p may function as an oncogene in osteosarcoma development and it may also provide a therapeutic strategy for controlling osteosarcoma progression.