沉默circZFR通过调控miR-497-5p表达抑制脊索瘤细胞的增殖、迁移及侵袭

Silence of circZFR expression inhibit the proliferation,migration and invasion of chordoma cells by regulating the expression of miR-497-5p

目的:研究环状RNA(circular RNAs,circRNAs)circZFR是否通过调控微小RNA(microRNA,miRNA/miR)-497-5p的表达影响脊索瘤细胞增殖、迁移及侵袭。方法:实时荧光定量PCR(quantitative real-time polymerase chain reaction,qRT-PCR)检测脊索瘤组织中circZFR和miR-497-5p的表达。在脊索瘤U-CH1细胞中转染si-circZFR或miR-497-5p。噻唑蓝(methyl thiazolyl tetrazolium,MTT)检测细胞增殖,Transwell小室法检测细胞迁移及侵袭情况,蛋白质印迹法(Western blot)检测细胞周期蛋白D1(CyclinD1)、p21、基质金属蛋白酶-2(matrix metalloprotease-2,MMP-2)、基质金属蛋白酶-9(matrix metalloprotease-9,MMP-9)蛋白表达,StarBase预测工具和双荧光素酶报告实验分析circZFR与miR-497-5p的靶向结合。si-circZFR和anti-miR-497-5p共转染,观察下调miR-497-5p表达对沉默circZFR表达诱导的U-CH1细胞增殖、迁移及侵袭的作用。结果:与髓核组织比较,脊索瘤组织中的circZFR表达量明显增加,miR-497-5p表达量显著减少(P<0.05)。沉默circZFR表达或过表达miR-497-5p明显降低U-CH1细胞48 h、72 h的细胞活性、迁移细胞数、侵袭细胞数、CyclinD1、MMP-2、MMP-9蛋白表达量,显著提高p21蛋白水平(P<0.05)。circZFR靶向调控miR-497-5p的表达。下调miR-497-5p表达逆转了沉默circZFR表达对脊索瘤U-CH1细胞增殖、迁移、侵袭、CyclinD1、MMP-2及MMP-9蛋白表达的抑制作用,和对p21蛋白表达的促进作用。结论:沉默circZFR表达可以抑制脊索瘤细胞的增殖、迁移及侵袭,其作用机制与靶向调控miR-497-5p的表达有关。

Objective:To investigate whether circular RNAs(circRNAs)circZFR affects the proliferation,migration and invasion of chordoma cells by regulating the expression of microRNA(miRNA/miR)-497-5p.Methods:The expression of circZFR and miR-497-5p in chordoma tissues was detected by real-time fluorescent quantitative PCR(qRT-PCR).si-circZFR or miR-497-5p were transfected into chordoma U-CH1 cells.Methyl thiazolyl tetrazolium(MTT)was used to detect cell proliferation.Transwell chamber was applied to determine cell migration and invasion,and Western blot was employed to detect CyclinD1,p21,matrix metalloproteinase-2(MMP-2),and matrix metalloproteinase-9(MMP-9)protein expression.StarBase prediction tool and dual luciferase reporter assay analyzed the targeted binding of circZFR to miR-497-5p.si-circZFR and anti-miR-497-5p were co-transfected,and the effects of down-regulating miR-497-5p expression on the inhibition of circZFR expression-induced U-CH1 cell proliferation and migration were observed.Results:Compared with nucleus pulposus,the expression of circZFR in chordoma was significantly increased,and the expression of miR-497-5p was obviously reduced(P<0.05).Silence of circZFR expression or miR-497-5p overexpression dramatically reduced the cell viability at 48 h and 72 h,number of migrating cells,number of invading cells,and expression of CyclinD1,MMP-2,and MMP-9 proteins in U-CH1 cells,evidently increased p21 protein level(P<0.05).circZFR targeted the regulation of miR-497-5p expression.Down-regulating the expression of miR-497-5p reversed the silence of circZFR expression on chordoma U-CH1 cell proliferation,migration and invasion,CyclinD1,MMP-2,MMP-9 protein expression,and the promotion of p21 protein expression.Conclusion:Silence of circZFR expression could inhibit the proliferation,migration and invasion of chordoma cells,and its mechanism is related to the targeted regulation of miR-497-5p expression.