Sui Jing, Fu Yanyun, Zhang Yanqiu, Ma Shumei, Yin Lihong, Pu Yuepu, Liang Geyu
Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing 210009, China.
Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing 210009, China.
Chem Biol Interact. 2018 Jan 25;280:77-85. doi: 10.1016/j.cbi.2017.12.020. Epub 2017 Dec 13.
This study investigated the role of microRNA(miRNA) in regulating the cytotoxicity of TiO nanoparticles (nano-TiO) to RAW264.7 cells. RAW264.7 cells were treated with 0 and 100 μg/ml nano-TiO for 24 h (for miRNA analysis). The differentially expressed miRNAs were detected using Illumina HiSeq™ 2000 sequencing. Through the bio-informatics analysis, miR-350 was found to play an important role in multiple signaling pathways, including MAPK signaling pathway, NF-kappa B signaling pathway and Apoptosis. To characterize the miR-350 function, miR-350 mimic was transfected into RAW264.7 cells for 24 h. MTT and Flow Cytometry were performed to detect cell proliferation, apoptosis and cell cycle (repetition), respectively. QRT-PCR, Western Blot methods and Luciferase assays were applied to detect expression of putative target gene PIK3R3. The results showed that miRNA profiles were differentially dysregulated. The apoptosis rate of miR-350 mimic group was significantly higher than negative control group (p < .05). Cell proliferation and cell cycle had no significant differences between treatment and negative control group. Compared with negative control, the level of protein of PIK3R3 was significantly decreased (p < .05), and the expression of 3'UTR constructs of PIK3R3 was significantly decreased (p < .05) in miR-350 mimic group. The expression of miRNAs was changed after exposed to nano-TiO, and biological function and target gene results showed miR-350 may promote RAW264.7 cell apoptosis through the negative regulation of PIK3R3 gene. Our results could provide a basis for further understanding of toxicity and possible mechanisms of nano-TiO exposure.
本研究调查了微小RNA（miRNA）在调节二氧化钛纳米颗粒（纳米TiO）对RAW264.7细胞细胞毒性中的作用。将RAW264.7细胞分别用0和100μg/ml纳米TiO处理24小时（用于miRNA分析）。使用Illumina HiSeq™ 2000测序检测差异表达的miRNA。通过生物信息学分析，发现miR-350在包括丝裂原活化蛋白激酶（MAPK）信号通路、核因子κB（NF-κB）信号通路和凋亡在内的多个信号通路中起重要作用。为了表征miR-350的功能，将miR-350模拟物转染到RAW264.7细胞中24小时。分别进行MTT法和流式细胞术检测细胞增殖、凋亡和细胞周期（重复实验）。应用实时定量聚合酶链反应（QRT-PCR）、蛋白质免疫印迹法和荧光素酶报告基因检测法检测假定靶基因PIK3R3的表达。结果显示，miRNA谱存在差异失调。miR-350模拟物组的凋亡率显著高于阴性对照组（p<0.05）。处理组与阴性对照组之间的细胞增殖和细胞周期无显著差异。与阴性对照组相比，miR-350模拟物组中PIK3R3的蛋白水平显著降低（p<0.05），PIK3R3的3'非翻译区（3'UTR）构建体的表达显著降低（p<0.05）。暴露于纳米TiO后miRNA表达发生变化，生物学功能和靶基因结果表明miR-350可能通过负调控PIK3R3基因促进RAW264.7细胞凋亡。我们的结果可为进一步了解纳米TiO暴露的毒性及可能机制提供依据。
