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

Objective To investigate the effects of paraquat on microRNA 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 realtime 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 PCl2 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＜0.01).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. Key words: Paraquat; PC12 cells; Microarray; Proto-oncogene proteins c-bcl-2