摘要
目的探讨锰处理SHSY5Y细胞中miRNA的差异表达及其相关靶基因表达的影响。方法:以500μmol/L MnCl2处理SHSY5Y细胞0h、2h、4h、6h、8h后用MTT检测细胞活力;选取500μmol/LMnCl26 h处理组与对照组分别进行miRNA芯片检测,分析差异表达的miRNA差异倍数(FC≥2.0且P≤0.05);挑选个差异表达的miRNA(miR-138-5p、miR-29a-3p),通过TargetScan,PITA,microRNAorg数据库对差异miRNA进行靶基因预测,取交集用于靶基因的筛选;用RT-PCR和western分别检测靶基因SIRT1基因和蛋白表达情况。结果:500μmol/L MnCl_2处理SHSY5Y细胞后,细胞毒性呈时间依赖性,时间越长细胞毒性越明显。miRNA芯片结果显示,与对照组相比,处理组中miR-138-5p和miR-29a-3p表达下调,差异有统计学意义(P<0.05),此结果也在定量即时聚合酶链锁反应(qRT-PCR)试验中得到了验证。同时,我们使用qRT-PCR和免疫印迹试验(Western Blot)发现靶基因SIRT1基因mRNA水平和蛋白水平的表达上调。结论:锰能够引起SHSY5Y细胞miR-138-5p、miR-29a-3p表达下调及相关靶基因SIRT1表达上调。SIRT1基因表达上调与细胞自噬作用密切相关,因此miR-138-5p、miR-29a-3p可能通过靶向作用于SIRT1基因影响锰诱导的SHSY5Y细胞自噬。
Objective To explore the expression of miR-138-5p,miR-29a-3p in SHSY5 Y cell induced by manganese.Methods SHSY5 Y cells were treated with 500 μmol/L MnCl_2·4H_2O,cell activity was tested by MTT assay;microarray analysis to show alterations of miRNA expression in an in SHSY5 Y cell.By TargetScan,PITA,microRNAorg database to predict the target genes,take the intersection for the Screening;RT-PCR and western blot were evaluated the expression of SIRT1.Results MnCl_2-4H_2O could time-dependently suppress the activity of SHSY5 Y cell;The microarray results reveal that treatment with500μM MnCl_2-4H_2O caused miRNA deregulation or upregulated;Alterations in the expressions of miR-138-5p,miR-29a-3p were verified by real-time quantitative reverse transcriptase polymerase chain reaction;.The results show miR-138-5pand miR-29a-3p deregulation.RT-PCR and western blot were evaluated the expression of SIRT1 was upregulated.Conclusion MnCl_2:4H_2O could cause the SHSY5 Y cell miR-138-5p and miR-29a-3p deregulation and upregulated the predicted target gene of SIRT1.Multiple evidence indicates SIRT1 promotes autophagy.miR-138-5p and miR-29a-3p may regulates manganese induced autophagy through targeting of SIRT1.
引文