摘要
目的 探讨长链非编码RNA(lncRNA)OPA相互作用蛋白5反义转录本1(OIP5-AS1)对脑胶质瘤细胞增殖、凋亡、迁移和侵袭的影响机制。方法 收集33例胶质瘤患者(低级别胶质瘤14例、高级别胶质瘤19例)和33例颅脑损伤患者的组织标本。实时荧光定量PCR(qPCR)检测组织和细胞中OIP5-AS1、微小RNA-942-5p(miR-942-5p)和检查点激酶1(CHEK1)mRNA表达,分析胶质瘤组织中OIP5-AS1、miR-942-5p和CHEK1 mRNA表达水平的相关性。体外培养人脑胶质瘤细胞系U87、SHG-44、U251、H4和正常人星形胶质细胞NHA,Western blot检测细胞中CHEK1蛋白表达。将U87细胞分为对照(NC)组、siRNA阴性对照(si-NC)组、OIP5-AS1 siRNA(si-OIP5-AS1)组、siOIP5-AS1+inhibitor阴性对照(si-OIP5-AS1+anti-NC)组、si-OIP5-AS1+miR-942-5p抑制剂(si-OIP5-AS1+anti-miR-942-5p)组,采用Lipofectamine 3000试剂进行转染。转染后,qPCR和Western blot检测细胞中OIP5-AS1、miR-942-5p和CHEK1 mRNA和蛋白表达水平;MTT法测定细胞增殖活性;流式细胞术检测细胞凋亡;Transwell实验检测细胞迁移和侵袭能力。最后通过双荧光素酶和RNA免疫沉淀(RIP)实验验证OIP5-AS1和miR-942-5p以及CHEK1和miR-942-5p的相互作用。结果 OIP5-AS1和CHEK1在脑胶质瘤组织和细胞中过表达,miR-942-5p呈低表达(均P<0.05);相关分析显示,脑胶质瘤组织中OIP5-AS1与miR-942-5p的表达水平呈负相关,miR-942-5p与CHEK1mRNA的表达水平呈负相关,CHEK1与OIP5-AS1 mRNA的表达水平呈正相关;且OIP5-AS1、CHEK1 mRNA在高级别胶质瘤组织中的表达明显高于低级别组织,而高级别胶质瘤中的miR-942-5p水平明显低于低级别组织(P<0.01)。沉默OIP5-AS1可显著上调miR-942-5p,抑制CHEK1的mRNA和蛋白表达(均P<0.05);沉默OIP5-AS1可显著抑制U87细胞增殖、迁移和侵袭,促进U87细胞凋亡(均P<0.05);下调miR-942-5p可上调CHEK1表达,阻断OIP5-AS1沉默对脑胶质瘤细胞生物学行为的影响(均P<0.05)。双荧光素酶和RIP实验证实miR-942-5p是OIP5-AS1的靶基因,CHEK1是miR-942-5p的下游靶基因。结论 沉默OIP5-AS1可能通过上调miR-942-5p抑制CHEK1表达,抑制脑胶质瘤细胞的侵袭和迁移,并促进细胞凋亡。
Objective To investigate the influence of long non-coding RNA(lncRNA) OPA-interacting protein 5 antisense transcript 1(OIP5-AS1) on the proliferation,apoptosis,migration and invasion of glioma cells.Methods Tissue specimens from 33 glioma patients(14 low-grade gliomas and 19 high-grade gliomas) and 33 patients with craniocerebral injury were collected.Real-time quantitative PCR(qPCR) was used to detect the mRNA expression levels of OIP5-AS1,microRNA-942-5p(miR-942-5p) and checkpoint kinase 1(CHEK1) in tissue samples and cells,and analyze the correlation between OIP5-AS1,miR-942-5p and CHEK1 mRNA expression level in glioma tissues.Human glioma cell lines U87,SHG-44,U251 and H4 and normal human astrocytes NHA were cultured in vitro.The expression of CHEK1 protein in cells were detected by Western blot assay.U87 cells were divided into the control(NC) group,the siRNA negative control(si-NC) group,the OIP5-AS1 siRNA(si-OIP5-AS1) group,the si-OIP5-AS1+inhibitor negative control(si-OIP5-AS1+anti-NC) group and the si-OIP5-AS1+miR-942-5p inhibitor(si-OIP5-AS1+anti-miR-942-5p) group.Cells were transfected with Lipofectamine 3000 reagent.After transfection,the expression levels of OIP5-AS1,miR-942-5p and CHEK1 mRNA and protein in cells were detected by qPCR and Western blot assay.MTT assay was used to detect the cell proliferation activity.Flow cytometry was used to detect cell apoptosis.Transwell assay was used to detect cell migration and invasion abilities.Finally,the interaction of OIP5-AS1 and miR-942-5p and CHEK1 and miR-942-5p were verified by dual luciferase and RNA immunoprecipitation(RIP) experiments.Results OIP5-AS1 and CHEK1 were overexpressed in glioma tissue and cells,while miR-942-5p was underexpressed(all P <0.05).Correlation analysis showed that OIP5-AS1 was negatively correlated with miR-942-5p,and miR-942-5p and CHEK1 mRNA was negatively correlated in glioma tissue.The expression level of CHEK1 mRNA was positively correlated with OIP5-AS1.The expression levels of OIP5-AS1 and CHEK1 mRNA were significantly higher in high-grade glioma tissue than those in low-grade glioma tissue,while the expression level of miR-942-5p was significantly lower in high-grade glioma tissue than that in low-grade glioma tissue(P <0.01).Silencing OIP5-AS1 significantly up-regulated miR-942-5p and inhibited the mRNA and protein expression of CHEK1(P <0.05).Silencing OIP5-AS 1 significantly inhibited the proliferation,migration and invasion of U87 cells,promoting apoptosis of U87 cells(P <0.05).Down-regulation of miR-942-5p could up-regulate the expression of CHEK1 and block the effects of OIP5-AS1 silencing on glioma cell proliferation,migration,invasion and apoptosis(P<0.05).Dualluciferase and RIP experiments confirmed that miR-942-5p was a target of OIP5-AS 1,and CHEK1 was a downstream target gene of miR-942-5p.Conclusion Silencing OIP5-AS1 may inhibit the expression of CHEK1 by up-regulating miR-942-5p,inhibit the invasion and migration of glioma cells,and promote cell apoptosis.
作者
陈明武
王开宇
杨波
郑诗豪
CHEN Mingwu;WANG Kaiyu;YANG Bo;ZHENG Shihao(Department of Neurosurgery,Fujian Provincial Hospital,Fuzhou 350001,China)
出处
《天津医药》
CAS
北大核心
2022年第12期1246-1253,共8页
Tianjin Medical Journal
基金
福建省自然科学基金资助项目(2020J05269)。
