百草枯诱导PC12细胞MicroRNA的变化及bcl-2调控机制

Micro RNA alteration after paraquat induced PC12 cells damage and regulatory mechanism of bcl-2

目的探讨百草枯（paraquat，PQ）诱导大鼠肾上腺嗜铬细胞瘤细胞（PCI2）细胞毒性中MicroRNA（miRNA）的表达及bcl-2的调控机制。方法以PCI2细胞为多巴胺能神经元的细胞模型，用终浓度为0、62．5μmol/LPQ分别诱导PCI2细胞24h，用微列阵芯片技术筛选变化显著的miRNA，实时荧光定量反转录一聚合酶链反应（realtimePCR）进行验证；根据microRNA数据库（TargetScan5．1）提供的靶标预测分析结果，搜寻其相关的靶标；用终浓度为0、62．5、125．0、250．0、500．0、1000．0μmol／LPQ染毒细胞24h，Annexin V-FITC／PI法流式细胞仪测定细胞凋亡，RT-PCR测定miR．34a、miR-Let-7e的相对表达水平，免疫印迹法（Western blot）测定bcl-2蛋白表达水平。结果与对照组比较，125．0、250．0、500．0、1000．0μmol／LPQ染毒PCI2细胞24h后，细胞存活率明显降低，细胞凋亡率明显上升，差异均有统计学意义（P〈0．05或P〈0．01），且呈现剂量依赖性。基因芯片技术显示，62．5μmol／LPQ作用于PCI2细胞24h引起8个miRNA表达下调，11个miRNA表达上调；其中miR-34a上调、miR-Let-7e下调最为明显。RT-PCR显示，随着PQ浓度的增加，miR-Let-7e表达水平持续下降，miR-34a表达水平持续上升，与芯片结果一致；随着PQ染毒剂量增加，bcl-2蛋白表达水平明显降低，且与miR-34a的表达量呈剂量一反应关系。结论miRNAs的异常表达参与了PQ致PCI2细胞损伤过程。PQ可能通过上调miR-34a表达增加bcl-2蛋白表达，进而诱导PCI2细胞凋亡。

Objective ;Fo investigate the effects of paraquat on mieroRNA expressions in PC12 cells, and to explore the regulatory mechanism of bcl-2. Methods We used PC12 cells as a popular in vitro cell model system for characterizing the dopaminergic neuron. After 24 h treatment with different concentrations of PQ （0, 62.5 μmol/L）, expression difference of microRNA was detected by microarray and examined by real- time quantitative PCR （RT-PCR）. Cell apoptosis was analyzed with flow cytometry （FCM） and the relative levels of miR-34a, miR-Let-7e were measured by RT-RCR following the PC12 cells treatment with 0, 62.5, 125, 250, 500, 1000 μmol/L PQ. Meanwhile, the protein expression of bcl-2 was evaluated by western blot according to forecasting targets analysis databases. Results Cell viability decreased and cell apoptosis increased with increasing PQ concentrations （from 125 to 1000 μmol/L） in a dose-dependent manner （P〈0.05 or P〈0.01）. MiRNA microarray showed that after 62.5 μmol/L PQ treatment, 11 miRNAs were significantly up-regulated while 8 miRNAs were down-regulated compared with control （P〈O.O1）. We chose miR-34a, miR-Let-7e which appeared most remarkable changes in microarray to examine by RT-PCR. It revealed that the level of miR-34a gradually ascended while miR-Let-7e declined after PQ treatment,which are accordant to the microarray results. The protein expression of bcl-2 treated with PQ significantly decreased compared with control and presented a negative correlation with the expression of miR-34a （P〈0.05 or P〈0.01）. Conclusion The alteration of miRNAs expression may be involved in the neurotoxicity of PQ. Especially, mir-34a negatively regulated the level of bcl-2, and thus plays a key role in PQ-induced cell apoptosis.