Huang M, Cai Q, Li H H, Lou D
School of Public Health, Ningxia Medical University, Yinchuan 750004, China.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2017 Jan 20;35(1):19-24. doi: 10.3760/cma.j.issn.1001-9391.2017.01.005.
To investigate the impacts of paraquat on microRNA profiles in apoptosis of human neural progenitor cells (hNPCs) and to explore miRNA targets and biological functions. We used hNPCs as a popular in vitro cell model system for characterizing the neurotoxicity. Cell apoptosis was detected by Annexin V-APC/7-AAD after 24 h treatment with different concentrations of PQ (0, 5, 10, 20, 40, 80 μmol/L). Microarray profiling expression of PQ treated cell line and their corresponding control was determined and differentially expression miRNAs were confirmed by quantitatively real-time PCR. The target genes regulated by aberrantly expressed miRNAs were predicted by on line-available software (Target Scan, Miranda, Mirbase). The GO and KEGG pathway database were used to analyze the functions of target genes. Meanwhile, the apoptosis-related protein expressions were evaluated by western blot. cell apoptosis increased with increasing PQ concentrations (from 10 to 80μmol/L) in a dose-dependent manner (<0.05). miRNA microarray showed that 40 miRNAs were significantly up-regulated while 26 miRNAs were down-regulated after 20 μmol/L PQ treatment (< 0.05). We selected 6 differentially expressed miRNAs to validate with qRT-PCR. The results were consistent with microarray data for all miRNAs tested. Bioinformatics analysis demonstrated target genes were enriched in regulation of neuron apoptosis and differentiation, MARK signaling pathway as well as P53 signaling pathway. The protein expressions of bax and caspase3 significantly increased while bcl-2 significantly decreased treated with PQ compared with control group (<0.05). There is a specific miRNA expression profile in paraquat-induced apoptosis of hNPCs. Differentially expression miRNAs regulated apoptosis of hNPCs through multiple molecular signaling pathways and especially for mitochondrial apoptosis pathway.
研究百草枯对人神经祖细胞（hNPCs）凋亡过程中微小RNA（miRNA）谱的影响，并探索miRNA的靶标和生物学功能。我们使用hNPCs作为一种常用的体外细胞模型系统来表征神经毒性。用不同浓度的百草枯（PQ，0、5、10、20、40、80μmol/L）处理24小时后，通过膜联蛋白V-藻红蛋白/7-氨基放线菌素D检测细胞凋亡。测定PQ处理细胞系及其相应对照的基因芯片表达谱，并通过定量实时PCR确认差异表达的miRNA。通过在线可用软件（Target Scan、Miranda、Mirbase）预测异常表达的miRNA调控的靶基因。利用基因本体论（GO）和京都基因与基因组百科全书（KEGG）通路数据库分析靶基因的功能。同时，通过蛋白质印迹法评估凋亡相关蛋白的表达。随着PQ浓度从10μmol/L增加到80μmol/L，细胞凋亡呈剂量依赖性增加（P<0.05）。miRNA芯片显示，20μmol/L PQ处理后，40种miRNA显著上调，26种miRNA下调（P<0.05）。我们选择6种差异表达的miRNA进行qRT-PCR验证。所有测试miRNA的结果与芯片数据一致。生物信息学分析表明，靶基因富集于神经元凋亡和分化调控、丝裂原活化蛋白激酶（MAPK）信号通路以及P53信号通路。与对照组相比，PQ处理后bax和caspase3的蛋白表达显著增加，而bcl-2显著降低（P<0.05）。在百草枯诱导的hNPCs凋亡中存在特定的miRNA表达谱。差异表达的miRNA通过多种分子信号通路调控hNPCs的凋亡，尤其是线粒体凋亡通路。
