miR-422a通过ATG12调控骨肉瘤细胞自噬及凋亡的研究

目的探讨miR-422a调控骨肉瘤细胞自噬及凋亡的作用和机制及其与靶基因ATG12的关系。方法采用瞬时转染miR-422a模拟物和抑制物分别上调或下调骨肉瘤细胞的miR-422a表达水平,采用MTT法检测各时间点骨肉瘤细胞的增殖能力,瞬时转染miR-422a后采用流式细胞术检测骨肉瘤细胞凋亡情况,采用实时荧光定量聚合酶链式反应(RT-qPCR)检测转染miR-422a对靶基因mRNA的影响。结果RT-qPCR结果显示,miR-422a在肿瘤组织及细胞中均高表达,ATG12在肿瘤组织及细胞中均低表达。MTT法结果显示,miR-422a-mimic组吸光度值明显升高,miR-422a-inhibitor组吸光度值明显降低(P<0.05)。流式细胞术检测结果显示,miR-422a-inhibitor组细胞凋亡率高于空白组与NC组(P<0.0001)。Western blot检测结果显示,miR-422a-mimic组细胞中ATG12、LC3-Ⅱ和LC3-Ⅰ蛋白量明显低于空白组与NC组(P<0.05);miR-422a-inhibitor组细胞中ATG12、LC3-Ⅱ蛋白量明显高于空白组与NC组,LC3-Ⅰ蛋白量明显低于空白组与NC组(P<0.05);miR-422a-inhibitor组细胞中LC3-Ⅱ/Ⅰ明显高于空白组与NC组(P<0.05)。结论miR-422a/ATG12信号轴可能调控骨肉瘤细胞的自噬及凋亡。

Study of miR-422a regulates autophagy and apoptosis of osteosarcoma cells through ATG12

Objective To investigate the role and mechanism of miR-422 a in regulating autophagy and apoptosis of osteosarcoma cells and its relationship with target gene ATG12.Methods The miR-422 a expression level was up-regulated or down-regulated by transient transfection with miR-422 a mimic and inhibitor,respectively.Proliferation ability of osteosarcoma cells at each time point was determined by MTT method.Flow cytometry was used to detect the apoptosis of osteosarcoma cells after transient transfection with miR-422 a.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the influence of transfection miR-422 a on target gene mRNA.Results The RT-qPCR results showed that miR-422 a highly expressed in tumor tissues and cells,while ATG12 low expressed in tumor tissues and cells.Result of MTT method showed that the absorbance value of the miR-422 a-mimic group significantly increased,while that of the miR-422 a-inhibitor group significantly decreased(P<0.05).The results of flow cytometry showed that the apoptosis rate of the miR-422 a-inhibitor group were higher than that of the blank group and the NC group(P<0.0001).The Western blot results showed that the protein levels of ATG12,LC3-Ⅱand LC3-Ⅰin the miR-422 a-mimic group were significantly lower than those in the blank group and the NC group(P<0.05);the protein levels of ATG12 and LC3-Ⅱin the miR-422 a-inhibitor group were significantly higher than those in the blank group and the NC group,and the protein level of LC3-Ⅰwas significantly lower than that in the blank group and the NC group(P<0.05);the LC3-Ⅱ/Ⅰin the miR-422 a-inhibitor group was significantly higher than that in the blank group and the NC group(P<0.05).Conclusion The miR-422 a/ATG12 signal axis may regulate autophagy and apoptosis of osteosarcoma cells.