环状RNA_3885通过吸附微小RNA-142-3p增强细胞周期蛋白依赖性激酶4功能提高骨肉瘤细胞的增殖与侵袭能力

Circular RNA-3885 enhances Cyclin dependent kinase 4 function through adsorption of microRNA-142-3p to improve proliferation and invasion of osteosarcoma cells

目的探讨环状RNA(circRNA,circ)_3885对骨肉瘤细胞增殖与侵袭行为的影响,并初步分析其作用机制。方法体外培养骨肉瘤MG-63细胞,设空白对照组、序列对照组、微小RNA(miR)-142-3p过表达组、circ_3885过表达组及联合干预组,其中miR-142-3p过表达组加入miR-142-3p模拟物(miR-142-3p mimic)共培养,circ_3885过表达组加入circ_3885过表达质粒共培养,联合干预组同时加入miR-142-3p mimic与circ_3885过表达质粒,而序列对照组加入miR-142-3p mimic的对照序列mimic NC和circ_3885过表达质粒的对照质粒OE-NC共培养。共培养48 h,采用细胞计数试剂盒(CCK-8)实验测定细胞增殖活力,划痕实验和Transwell小室实验测定细胞迁移与侵袭能力;共培养7 d,采用蛋白质印迹法(Western blot)实验检测细胞周期蛋白依赖性激酶4(CDK4)和细胞周期蛋白(Cyclin)A与Cyclin E的表达。多组间的比较行单因素方差分析,事后两两比较采用LSD-t或SNK检验。结果空白对照组、序列对照组、miR-142-3p过表达组、circ_3885过表达组及联合干预组的增殖活力分别为0.852±0.095、0.878±0.080、0.705±0.080、1.125±0.122、0.985±0.087,细胞迁移率分别为(25.3±5.6)%、(24.0±6.3)%、(12.5±3.2)%、(66.7±13.5)%、(42.3±8.3)%,差异均有统计学意义(F=8.322、20.032,P均<0.05);其中miR-142-3p过表达组增殖活力与迁移数目低于序列对照组,circ_3885过表达组高于序列对照组;联合干预组高于miR-142-3p过表达组同时低于circ_3885过表达组(P<0.05)。空白对照组、序列对照组、miR-142-3p过表达组、circ_3885过表达组及联合干预组的CDK4表达水平分别为0.305±0.052、0.312±0.047、0.175±0.026、0.648±0.085、0.432±0.054,Cyclin A表达水平分别为0.274±0.032、0.266±0.045、0.105±0.020、0.584±0.074、0.385±0.052,差异均有统计学意义(F=30.195、41.224,P<0.001);其中miR-142-3p过表达组的CDK4与Cyclin A表达水平均低于序列对照组(P<0.05),circ_3885过表达组均高于对照组(P<0.05);联合干预组高于miR-142-3p过表达组同时低于circ_3885过表达组(P<0.05)。结论circ_3885能够作为miR-142-3p的分子海绵下调miR-142-3p表达,从而释放CDK4,增强骨肉瘤细胞侵袭能力。

Objective To investigate the effects of circular RNA(circRNA,circ)-3885 on the proliferation and invasion behavior of osteosarcoma cells,and to analyze its mechanism.Methods MG-63 cells were cultured in vitro,and divided into blank control group,sequence control group,microRNA(miR)-142-3p overexpression group,circ_3885 overexpression group and combined intervention group.The cells in miR-142-3p overexpression group were co-cultured with miR-142-3p mimic,co-cultured with circ_3885 overexpression plasmid in the circ_3885 overexpression group,and co-cultured with both in the combined intervention group.The cells in the control group were co-cultured with a control mimic NC of miR-142-3p mimic and a control plasmid OE-NC of circ_3885 overexpressed plasmid.After co-culture for 48 h,cell proliferation activity was determined by cell counting kit-8(CCK-8)assay,and cell migration and invasion ability was determined by scratch assay and Transwell assay.After 7 days of co-culture,the expression levels of Cyclin dependent kinase 4(CDK4),Cyclin A and Cyclin E were detected by Western blotting.The comparison between multiple groups was performed by one-way ANOVA,and the subsequent comparison between two groups was performed by LSD or SNK test.Results The proliferative activity of blank control group,sequence control group,miR-142-3p overexpression group,circ_3885 overexpression group and combined intervention group was 0.852±0.095,0.878±0.080,0.705±0.080,1.125±0.122,0.985±0.087,and the cell migration rates were(25.3±5.6)%,(24.0±6.3)%,(12.5±3.2)%,(66.7±13.5)%,(42.3±8.3)%,respectively.The differences were statistically significant(F=8.322,20.032,P<0.05).The proliferation activity and migration number of miR-142-3p overexpression group were lower than the sequence control group,and those of the circ_3885 overexpression group were higher than the sequence control group.The proliferation activity and migration number of the combined intervention group were higher than miR-142-3p overexpression group and lower than circ_3885 overexpression group(P<0.05).The expression levels of CDK4 in blank control group,sequence control group,miR-142-3p overexpression group,circ_3885 overexpression group and combined intervention group were 0.305±0.052,0.312±0.047,0.175±0.026,0.648±0.085,0.432±0.054,and those of Cyclin A were 0.274±0.032,0.266±0.045,0.105±0.020,0.584±0.074 and 0.385±0.052,respectively.The differences were statistically significant(F=30.195,41.224,P<0.05).The expression levels of CDK4 and Cyclin A in miR-142-3p overexpression group were lower than those in sequence control group,and those in circ_3885 overexpression group were higher than the sequence control group(P<0.05).The expression levels of CDK4 and Cyclin A in the combined intervention group were higher than miR-142-3p overexpression group and lower than circ_3885 overexpression group(P<0.05).Conclusion The circ_3885 can act as a molecular sponge of miR-142-3p to down-regulate the expression of miR-142-3p,thereby releasing CDK4 and enhancing the invasion ability of osteosarcoma cells.