干扰苹果酸酶3对人脑胶质瘤细胞线粒体分裂与融合的影响

The roles of malic enzyme 3 knockdown in mitochondrial fusion and fission in human glioma cells

目的:研究苹果酸酶3(malic enzyme 3,ME3)对人脑胶质瘤细胞线粒体分裂与融合的影响。方法:将干扰质粒sh-ME3或对照质粒sh-EGFP转染到人脑胶质瘤LN229细胞中,用实时定量PCR、蛋白质印迹法检测干扰效率。蛋白质印迹法检测线粒体的融合与分裂指标及细胞凋亡相关蛋白;用荧光探针检测线粒体膜电位。结果:与对照组相比,实验组ME3的mRNA表达水平下降了76%(t=20.16,P<0.01),蛋白表达也相应减少。转染干扰质粒sh-ME3后,线粒体分裂相关的线粒体分裂蛋白1(mitochondrial fission 1 protein,Fis-1)和动力相关蛋白1(dynamin-related protein 1,DRP1)表达增高,而线粒体融合相关的线粒体融合蛋白1(mitofusin 1,Mfn1)、线粒体融合蛋白2(mitofusin 2,Mfn2)、视神经萎缩症蛋白1(optic atrophy 1,OPA1)表达降低;线粒体膜电位降低;细胞凋亡相关的水解型半胱氨酸蛋白酶(cleaved-caspase-3)表达增加,而B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)表达降低。结论:干扰ME3促进了胶质瘤细胞线粒体的分裂,抑制了其融合,降低了膜电位,从而引起细胞凋亡。

Objective:To investigate the effects of malic enzyme 3(ME3)on the ability of mitochondrial fusion and fission in human glioma cells.Methods:The sh-ME3 plasmids and sh-EGFP(control plasmids)were transfected into the glioma cell LN229.The plasmid knockdown efficiency were detected by real time PCR and Western blotting.Western blotting was used to detect the markers of apoptosis and mitochondrial fusion and fission,and membrane potential of mitochondria was detected by fluorescent probe.Results:Compared with the control group,the mRNA expression level of ME3 in the experimental group decreased by 76%(t=20.16,P<0.01),and the protein expression level also decreased;the mitochondrial fission related markers Fis-1 and DRP1 were increased,while the mitochondrial fusion related markers Mfn1,Mfn2,OPA1 were decreased,membrane potential of mitochondria was decreased;the apoptosis related marker cleaved-caspase 3 was increased,while Bcl-2 was decreased.Conclusion:The knockdown of ME3 promotes mitochondrial fission in glioma cells,inhibits their fusion,reduces membrane potential of mitochondria,thereby causes apoptosis.