右美托咪定通过miR-760/HDGF信号通路影响胶质瘤细胞的恶性生物学行为

Dexmedetomidine affects the malignant biological behavior of glioma cells via miR-760/HDGF

目的:探讨右美托咪定对胶质瘤细胞的恶性生物学行为的影响及其机制。方法:体外培养人脑胶质瘤细胞A172,用不同浓度(50、100、200、400、800 mg/L)的右美托咪定处理。采用甲基噻唑基四唑(MTT)法检测右美托咪定对A172细胞增殖能力的影响;流式细胞术检测右美托咪定对A172细胞凋亡能力的影响;Transwell小室实验检测右美托咪定对A172细胞迁移及侵袭能力的影响。实时定量PCR(qRT-PCR)与蛋白免疫印迹法(Western blot)分别检测右美托咪定对微小RNA-760(miR-760)、肝癌衍生生长因子(HDGF)表达的影响;双荧光素酶报告实验检测miR-760过表达对野生型和突变型HDGF荧光素酶活性的影响;A172细胞中转染miR-760 mimics及miR-con,检测细胞增殖、凋亡、迁移及侵袭能力的变化。Western blot检测基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved caspase-3)、细胞凋亡的标志蛋白PARP降解产物(Cleaved PARP)蛋白表达。结果:右美托咪定可明显降低胶质瘤细胞存活率(P<0.05),减少迁移细胞数与侵袭细胞数(P<0.05),抑制HDGF、MMP-2、MMP-9的表达(P<0.05),而提高细胞凋亡率(P<0.05),促进miR-760、Cleaved caspase-3、Cleaved PARP的表达(P<0.05),浓度达到800 mg/L时作用明显;荧光素酶报告基因结果证实miR-760能够抑制HDGF的3′-UTR区荧光素酶活性(P<0.05);转染miR-760 mimics后,与miR-con组比较,细胞存活率明显降低(P<0.05),迁移细胞数与侵袭细胞数明显减少(P<0.05),MMP-2、MMP-9的表达水平明显降低(P<0.05),而细胞凋亡率明显升高(P<0.05),Cleaved caspase-3、Cleaved PARP的表达水平明显升高(P<0.05);干扰miR-760的表达可逆转右美托咪定对胶质瘤细胞生物学行为的影响。结论:右美托咪定通过miR-760/HDGF信号通路减弱胶质瘤细胞增殖、迁移及侵袭能力,促进细胞凋亡。

Objective To investigate the effect of dexmedetomidine on the malignant biological behavior of glioma cells and its mechanism.Methods Human glioma cell line A172 was cultured in vitro and treated with different concentrations(50,100,200,400,800 mg/L)of dexmedetomidine.The effect of dexmedetomidine on the proliferation of A172 cells was detected by MTT.Flow cytometry was used to detect the effect of dexmedetomidine on the apoptosis of A172 cells.Transwell chamber assay was used to detect the effect of dexmedetomidine on the migration and invasion of A172 cells.The effects of dexmedetomidine on the expression of miR-760 and HDGF were detected by real-time quantitative PCR and Western blot.Dual luciferase reporter assays were used to examine the effect of miR-760 overexpression on wild-type and mutant HDGF luciferase activities.miR-760 mimics and miR-con were transfected into A172 cells to detect changes in cell proliferation,apoptosis,migration,and invasion.Western blot was used to detect the expression of MMP-2,MMP-9,Cleaved caspase-3,and Cleaved PARP protein.Results Dexmedetomidine significantly reduced the survival rate of glioma cells(P<0.05),decreased the number of migrated cells and the number of invasive cells(P<0.05),and inhibited the expression of HDGF,MMP-2,and MMP-9(P<0.05).However,it could increase the apoptosis rate(P<0.05)and promote the expression of miR-760,Cleaved caspase-3,and Cleaved PARP(P<0.05).The effect was obvious when the concentration reaches 800 mg/L.Luciferase reporter gene results confirmed that miR-760 can inhibit the luciferase activity of the 3'-UTR region of HDGF(P<0.05).After transfection of miR-760 mimics,cell viability was significantly lower than that of miR-con group(P<0.05),and the number of migrated cells and invasive cells were significantly decreased(P<0.05),and the expression levels of MMP-2 and MMP-9 were significantly decreased(P<0.05),but the apoptosis rate was significantly increased(P<0.05),and the expression levels of Cleaved caspase-3 and Cleaved PARP were significantly increased(P<0.05).Interference with the expression of miR-760 reversed the effect of dexmedetomidine on the biological behavior of glioma cells.Conclusion Dexmedetomidine can attenuate the proliferation,migration and invasion of glioma cells through miR-760/HDGF and promotes apoptosis.