微小RNA-494通过调节生存素表达对骨肉瘤凋亡和侵袭的调控作用

Effects of microRNA -494 on apoptosis and invasion against osteosarcoma cell through regulation of Survivin

目的观察微小RNA（miRNA，miR）-494通过调节生存素（Snrvivin）对骨肉瘤MG-63细胞增殖、凋亡及侵袭的影响。方法miR-494体外模拟物（mimic）转染，构建miR-494上调表达模型，实时定量聚合酶链反应（Real-time PCR）法验证转染效果；细胞计数试剂盒（CCK-8）法检测细胞增殖率变化；流式细胞技术检测细胞凋亡率变化；细胞体外侵袭试验检测细胞侵袭能力变化；Real-time PCR法和Western blot实验分别检测Survivin mRNA和蛋白表达水平。结果miR-494 mimic转染后，MG-63细胞中miR-494表达水平（4．38±0．53）较对照组（1．02±0．02）明显升高（t=6．312，P=0．003），miR-494mimic转染后24h及48h后细胞相对存活率均明显低于对照组，流式细胞仪结果显示mimic组细胞凋亡率[（8．76±0．82）％]较对照组[（5．43±0．63）％]明显提高（t=3．199，P=0．032），体外侵袭实验证实mimic组每视野下平均细胞数[（60．40±11．28）个]明显少于NC组[（109．51±7．46）个，t=3．628，P=0．022]，Real-time PCR和Western blot实验分别证实miR-494 mimic转染后，Survivin在mRNA和蛋白水平表达均明显抑制。结论miR-494表达上调可以抑制骨肉瘤细胞增殖和侵袭能力，诱导细胞凋亡，这些作用与下调Survivin表达有关。

Objective To investigate the effects of microRNA （miRNA, miR） -494 on the proliferation, apoptosis and invasion against osteosarcoma MG -63 cell through regulation of Survivin. Methods The expression of miR - 494 in osteosarcoma MG - 63 ceils was detected by real - time quantitative polymerase chain reaction （Real- time PCR） after miR- 494 mimic transfection. After the validation of miR - 494 in this model, cell count kit - 8 （ CCK - 8 ） and flow cytometry assay were used to detect the proliferation and apoptosis after miR -494 mimic transfection; Transwell assay was used to determine the invasion MG -63 cells. Then the expression levels of Survivin mRNA and proteins were detected by Real -time PCR and Western blotting respectively. Results After miR -494 mimic transfection in MG - 63 cells, relative expression of miR -494 in mimic group （4. 38 ± 0. 53 ） significantly increased compared with the negative - control （NC, 1.02 ±0. 02） group （t =6. 312, P =0. 003）. The proliferation 24 h and 48 h after miR -494 mimic transfection were significantly suppressed compared with the NC group. The flow cytometry assay suggested the apoptosis cell increased in mimic group （ 8.76 ± 0.82 ） % compared with the NC group [ （5.43 ±0. 63 ）%, t = 3. 199, P = 0. 032 ]. Transwell assay showed the invated cells significantly reduced in mimic group （60.40 ± 11.28） compared with the NC group （109. 51 ±7.46, t = 3. 628, P = 0. 022 ）. The expressions of Survivin in the mRNA and protein levels were reduced significantly in mimic group after transfection. Conclusion miR -494 inhibited the proliferation, invasion cell and induced apoptosis of osteosarcoma MG - 63 through the down - regulation of Survivin.