CacyBP/SIP介导的Parkin依赖型线粒体自噬对DA能神经元凋亡及细胞周期的调控作用

Regulation of CacyBP/SIP-mediated Parkin-dependent mitophagy on apoptosis and cycle of dopaminergic neurons

目的探讨钙周期素结合蛋白(CacyBP/SIP)介导的Parkin依赖型线粒体自噬对多巴胺(DA)能神经元凋亡及细胞周期的调控作用。方法将SH-SY5Y细胞分为模型组、对照组、CacyBP/SIP组, 其中模型组细胞用1-甲基-4-苯基吡啶(MPP+, 0.5 mmol/L)处理24 h, 对照组及CacyBP/SIP组细胞分别模型组细胞基础上进一步转染空载慢病毒或CacyBP/SIP-sgRNA慢病毒。采用Western blotting实验检测各组细胞中CacyBP/SIP、微管相关蛋白1轻链3(LC3)、溶酶体相关膜蛋白2(LAMP2)、磷酸酶张力蛋白诱导激酶1(Pink1)、Parkin、P53、Bcl-2、Bax的蛋白表达水平, 采用流式细胞仪检测各组细胞的凋亡水平及细胞周期情况, 采用免疫荧光单染法检测各组细胞中LC3、LAMP2的表达情况, 采用免疫荧光双染法检测各组细胞中CacyBP/SIP与Parkin的共表达情况。结果与模型组、对照组比较, CacyBP/SIP组CacyBP/SIP、LAMP2、Pink1、Parkin蛋白的表达明显减少, LC3-Ⅱ/Ⅰ值明显降低, LC3-Ⅱ、LAMP2的免疫荧光染色强度明显降低, Bcl-2蛋白表达明显减少, Bax蛋白表达明显增加, Bcl-2/Bax值明显降低, 细胞凋亡率明显提高, P53蛋白的表达明显增加, G1期细胞占比明显增加, CacyBP/SIP与Parkin共表达的免疫荧光染色强度明显降低, 差异均有统计学意义(P<0.05)。结论敲除CacyBP/SIP基因后, Parkin蛋白降低导致细胞阻滞于G1期, 并介导Parkin依赖型线粒体自噬功能降低, 从而导致细胞凋亡水平提高。

Objective To investigate the regulation of Parkin-dependent mitophagy mediated by calcyclin-binding protein and Siah-1 interacting protein(CacyBP/SIP)on apoptosis and cycle of dopaminergic(DA)neurons.Methods SH-SY5Y cells were divided into model group,control group and CacyBP/SIP group;cells in the model group were treated with 1-methyl-4-phenylpyridine(MPP+,0.5 mmol/L)for 24 h,and cells in the control group and CacyBP/SIP group were transfected with empty lentivirus or CacyBP/SIP-sgRNA lentivirus on the basis of MMP+(0.5 mmol/L)treatment for 24 h,respectively.Western blotting was used to detect the protein expression levels of CacyBP/SIP,microtubule-associated protein l light chain 3(LC3),lysosome-associated membrane protein 2(LAMP2),phosphatase and tensin homolog ten induced kinase 1(Pink1),Parkin,P53,Bcl-2,and Bax;flow cytometry was used to detect the cell apoptosis and cycle;immunofluorescent single staining was used to detect the expressions of LC3 and LAMP2;immunofluorescent double staining was used to detect the coexpressions of CacyBP/SIP and Parkin.Results As compared with the model group and control group,the CacyBP/SIP group had significant reduction in protein expressions of CacyBP/SIP,LAMP2,Pink1,and Parkin,LC3-II/I ratio,immunofluorescent staining intensities of LC3-II and LAMP2,and Bcl-2 protein expressions(P<0.05).As compared with the model group and control group,the CacyBP/SIP group had significantly increased Bax protein expression,significantly decreased Bcl-2/Bax ratio,significantly increased apoptosis rate,significantly increased P53 protein expression,significantly increased proportion of cells at G1 phase,and significantly decreased immunofluorescent intensity of CacyBP/SIP and Parkin co-expressions(P<0.05).Conclusion After knocking out CacyBP/SIP gene,the decrease of Parkin protein leads to cell cycle being arrested at G1 stage,and mediates the decrease of Parkin-dependent mitochondrial autophagy,thereby leading to increased cell apoptosis.