CircSEPT9调节miR-650/HSPB1轴对胶质瘤细胞恶性生物学行为影响的探讨

An investigation of the effect of CircSEPT9 on malignant biological behavior of glioma cells by regulating miR-650/HSPB1 axis

目的探讨环状RNA(CircRNA)SEPT9调节miR-650/热休克蛋白B1(HSPB1)轴对胶质瘤细胞恶性生物学行为的影响。方法通过实时定量聚合酶链式反应(qRT-PCR)、Western blot法检测胶质瘤组织、癌旁组织、人类正常神经胶质细胞HEB及胶质瘤细胞系LN18、SW1088、U251中CircSEPT9、miR-650、HSPB1蛋白表达水平。将U251细胞分为对照组(NC组)、si-NC组、si-CircSEPT9组、mimic NC组、miR-650 mimic组、si-CircSEPT9+inhibitor NC组、si-CircSEPT9+miR-650 inhibitor组、si-CircSEPT9+pcDNA组、si-CircSEPT9+pcDNA-HSPB1组。采用qRT-PCR法检测CircSEPT9、miR-650表达水平。采用Western blot法检测HSPB1、细胞周期蛋白D1(Cyclin D1)、Caspase-3、基质金属蛋白酶(MMP)-2、MMP-9蛋白表达水平。通过双荧光素酶基因实验验证CircSEPT9与miR-650、miR-650与HSPB1的关系。结果在胶质瘤组织和细胞中,CircSEPT9和HSPB1蛋白呈高表达,miR-650呈低表达,且在U251细胞中CircSEPT9以及HSPB1蛋白表达水平最高,miR-650表达水平最低,差异有统计学意义(P<0.05)。沉默CircSEPT9或过表达miR-650均可抑制U251细胞增殖、侵袭、迁移及Cyclin D1、MMP-2、MMP-9蛋白表达水平,促进细胞凋亡及Caspase-3蛋白表达。下调miR-650或过表达HSPB1均可减弱沉默CircSEPT9对U251细胞的作用影响。CircSEPT9与miR-650、miR-650与HSPB1存在靶向关系。结论沉默CircSEPT9可能通过海绵化上调miR-650来抑制HSPB1表达,进而抑制U251细胞增殖、迁移与侵袭,促进细胞凋亡。

Objective To investigate the effect of circular ribonucleic acid(CircRNA)septin 9(CircSEPT9)on malignant biological behavior of glioma cells by regulating micro ribonucleic acid-650(miR-650)/heat shock protein B1(HSPB1)axis.Methods The expressions of CircSEPT9,miR-650 and HSPB1 protein in glioma tissues,tumor-adjacent tissues,human normal glial cells HEB and glioma cell lines LN18,SW1088 and U251 were detected by quantitative real time polymerase chain reaction(qRT-PCR)and Western blot,respectively.U251 cells were divided into control group(NC group),si-NC group,si-CircSEPT9 group,mimic NC group,miR-650 mimic group,si-CircSEPT9+inhibitor NC group,si-CircSEPT9+miR-650 inhibitor group,si-CircSEPT9+pcDNA group,and si-CircSEPT9+pcDNA-HSPB1 group.The expression levels of CircSEPT9 and miR-650 were detected by qRT-PCR.Western blot was applied to detect the expression levels of HSPB1,Cyclin D1,Caspase-3,matrix metalloproteinase(MMP)-2 and MMP-9 proteins.Double luciferase gene experiment was applied to verify the relationship between CircSEPT9 and miR-650,[LM]and the relationship between miR-650 and HSPB1.Results In the glioma tissues and glioma cells,CircSEPT9 and HSPB1 were highly expressed,while miR-650 was low expressed.In the U251 cells,the expression levels of CircSEPT9 and HSPB1 were the highest,and the expression level of miR-650 was the lowest,and the differences were significant(P<0.05).Silencing CircSEPT9 or overexpressing miR-650 was able to inhibit U251 cell proliferation,invasion,migration and the expression levels of Cyclin D1,MMP-2 and MMP-9 proteins,and was able to promote cell apoptosis and Caspase-3 protein expression.The down-regulation of miR-650 or overexpression of HSPB1 could weaken the effect of CircSEPT9 silencing on the U251 cells.CircSEPT9 had a targeting relationship with miR-650,and miR-650 had a targeting relationship with HSPB1.Conclusion Silencing CircSEPT9 may inhibit the expression of HSPB1 by up-regulating miR-650 through spongification,thereby inhibiting the proliferation,migration and invasion of U251 cells and promoting apoptosis.