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Title: [Alterations of miRNA profiles and function analysis in paraquat-induced apoptosis of hNPCs].
Author: Huang M, Cai Q, Li HH, Lou D.
Journal: Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi; 2017 Jan 20; 35(1):19-24. PubMed ID: 28241697.
Abstract:
Objective: 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. Methods: 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. Results: cell apoptosis increased with increasing PQ concentrations (from 10 to 80μmol/L) in a dose-dependent manner (P<0.05). miRNA microarray showed that 40 miRNAs were significantly up-regulated while 26 miRNAs were down-regulated after 20 μmol/L PQ treatment (P< 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 (P<0.05). Conclusion: 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. 目的：观察百草枯（Paraquat，PQ）诱导人胚胎神经干细胞（human neural progenitor cells，hNSCs）凋亡过程中MicroRNA （miR ）表达谱的特征性变化，分析差异性表达miRNAs的靶基因及生物学信息。 方法：以永生化的人胚胎神经干细胞系（ReNcell CX细胞）为细胞模型，用终浓度为0、5、10、20、40和80 μmol/L PQ处理细胞24 h, Annexin V-APC/7-AAD法测定细胞凋亡率；用终浓度0、20 μmol/L PQ分别诱导细胞24 h，采用miRCURYrMLNA miRNA芯片技术筛选显著性差异表达的miRNA，实时荧光定量PCR （qRT-PCR）进行验证；根据TargetScan、Miranda、Mirbase 3个数据库对筛选出的差异表达的miRNA进行靶基因预测；利用Gene Ontology （GO）和KEGG Pathway数据库进行靶基因生物学信息分析；采用Western blot测定凋亡相关蛋白表达。 结果：与对照组比较，PQ (10、20、40、80 μmol/L）作用细胞后，细胞凋亡率明显升高，呈现剂量依赖方式，差异有统计学意义（P<0.05）；20 μmol/L PQ诱导细胞24 h，miRNA表达谱出现特征性变化，显著上调表达miRNAs 40个，下调表达miRNAs 26个（P<0.05）。选择6个差异表达的miRNA (miR-378b、miR-378d、miR-195-5p、miR-34c-5p、miR-145-3p、miR-124-3p）进行qRT-PCR验证，结果显示，芯片与qRT-PCR检测的结果趋势一致。生物学信息分析显示，差异表达miRNA调控的靶基因主要参与神经细胞凋亡、神经细胞分化、MARK信号通路、p53信号通路等。20、40、80 μmol/LPQ染毒能够诱导细胞的bcl-2蛋白表达下调，bax、caspase3蛋白表达上调，与对照组比较，差异有统计学意义（P<0.05 ）。 结论： PQ诱导hNSCs凋亡过程中出现miRNA表达谱的特征性改变，差异表达的miRNA通过参与多种分子信号途径调控hNSCs细胞存亡，尤其是线粒体凋亡通路可能涉及其中。.

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