依托咪酯通过调控LncRNA CDKN2B-AS1/miR-199a-5p表达抑制胶质瘤细胞增殖、迁移和侵袭

Etomidate inhibits proliferation, migration and invasion of glioma cells by regulating the expression of LncRNA CDKN2B-AS1/miR-199a-5p

目的探讨依托咪酯对胶质瘤细胞的增殖、迁移和侵袭的影响及其作用机制。方法用不同浓度的依托咪酯处理胶质瘤细胞U251,采用四甲基偶氮唑蓝(MTT)检测依托咪酯对U251细胞增殖能力的影响;Transwell实验检测依托咪酯对U251细胞迁移及侵袭的影响;实时荧光定量-聚合酶链反应(qRT-PCR)检测胶质瘤组织中长链非编码RNA CDKN2B-AS1(LncRNA CDKN2B-AS1)与微小RNA-199a-5p(miR-199a-5p)的表达;采用上述方法检测抑制CDKN2B-AS1的表达或miR-199a-5p过表达对U251细胞增殖、迁移侵袭的影响;双荧光素酶报告实验验证CDKN2B-AS1与miR-199a-5p的靶向调控关系;qRT-PCR检测依托咪酯对CDKN2B-AS1与miR-199a-5p表达的影响,CDKN2B-AS1过表达以此验证依托咪酯对U251细胞增殖、迁移、侵袭的作用;Western印迹检测细胞周期蛋白(Cyclin)D1、基质金属蛋白酶(MMP)-2、MMP-9、p21蛋白表达。结果与Con组相比,依托咪酯不同剂量组U251细胞抑制率显著升高,迁移与侵袭细胞数显著减少,CyclinD1、MMP-2、MMP-9表达水平显著降低,p21表达水平显著升高,且随着依托咪酯使用剂量的增加而显著变化;胶质瘤组织中CDKN2B-AS1的表达水平显著升高,而miR-199a-5p的表达水平显著降低(均P<0.05);抑制CDKN2B-AS1的表达或miR-199a-5p过表达能够抑制U251细胞增殖、迁移及侵袭;双荧光素酶报告实验证明CDKN2B-AS1可靶向结合miR-199a-5p而抑制其表达及活性(P<0.05);依托咪酯可显著提高miR-199a-5p的表达水平,降低CDKN2B-AS1的表达水平(均P<0.05);CDKN2B-AS1过表达可逆转依托咪酯对U251细胞增殖、迁移、侵袭的抑制作用。结论依托咪酯可通过调控CDKN2B-AS1/miR-199a-5p表达抑制胶质瘤细胞的增殖、迁移及侵袭。

Objective To explore the effects of etomidate on the proliferation,migration and invasion of glioma cells and its action mechanism.Methods The glioma cell U251 was treated with different concentrations of etomidate and the effect of etomidate on the proliferation of U251 cells was detected by MTT.The Transwell assay was used to examine the effect of etomidate on migration and invasion of U251 cells.qRT-PCR was used to detect the expression of LncRNA CDKN2B-AS1 and miR-199a-5p in glioma tissues.The above method was used to detect the effect of inhibiting the expression of CDKN2B-AS1 or the overexpression of miR-199a-5p on the proliferation and migration of U251 cells.The dual luciferase reporter assay verified the targeted regulatory relationship between CDKN2B-AS1 and miR-199a-5p.The effect of etomidate on the expression of CDKN2B-AS1 and miR-199a-5p was detected by qRT-PCR.The overexpression of CDKN2B-AS1 was used to verify the effect of etomidate on proliferation,migration and invasion of U251 cells.The expression of CyclinD1,MMP-2,MMP-9 and p21 protein was detected by Western blot.Results Compared with the Con group,the inhibition rate of U251 cells in different doses of etomidate was significantly increased(P<0.05),the number of migration and invasion cells was significantly decreased(P<0.05),and the expression levels of CyclinD1,MMP-2 and MMP-9 were significantly decreased(P<0.05),the expression level of p21 was significantly increased(P<0.05),and significantly changed with the increase of etomidate dose(P<0.05).The expression level of CDKN2B-AS1 in glioma tissues was significantly increased(P<0.05),while the expression level of miR-199a-5p was significantly decreased(P<0.05).Inhibition of CDKN2B-AS1 expression or miR-199a-5p overexpression inhibited U251 cell proliferation,migration and invasion.The dual luciferase reporter assay demonstrated that CDKN2B-AS1 could bind to miR-199a-5p and inhibit its expression and activity(P<0.05).Etomidate significantly increased the expression level of miR-199a-5p(P<0.05)and decreased the expression level of CDKN2B-AS1(P<0.05).Overexpression of CDKN2B-AS1 reversed the inhibitory effect of etomidate on proliferation,migration and invasion of U251 cells.Conclusions Etomidate inhibits the proliferation,migration and invasion of glioma cells by regulating the expression of CDKN2B-AS1/miR-199a-5p.