碳黑纳米颗粒诱导人支气管上皮细胞炎症反应中环状RNA的表达改变

Expression profile of circular RNA in inflammatory response in human bronchial epithelial cells induced by carbon black nanoparticles

目的探索碳黑纳米颗粒(CBNPs)对人支气管上皮细胞(16HBE细胞)的毒性效应,并根据全转录组高通量测序对差异表达的环状RNA进行筛选与鉴定,为CBNPs暴露的生物标志物研发与其在表观遗传毒理学的应用提供依据。方法于2020年6月,用20、40和80μg/ml浓度的CBNPs对16 HBE细胞进行染毒处理,以不加任何干预的16HBE细胞作为对照组,通过CCK8与乳酸脱氢酶(LDH)实验检测CBNPs细胞毒性。实时荧光定量PCR(qRT-PCR)及ELISA法检测各浓度CBNPs染毒72 h后白细胞介素-6(IL-6)和白细胞介素-8(IL-8)mRNA及蛋白水平改变,Western blot检测核因子-κB(NF-κB)相关炎性蛋白[Toll样受体4(TLR4)、磷酸化核因子-κB(P-NF-κB)、凋亡相关斑点样蛋白(ASC)和天冬氨酸蛋白水解酶1(Caspase-1)]的表达水平;根据高通量测序结果筛选并通过qRT-PCR鉴定差异表达的circRNA,选择稳定差异表达且与NF-κB通路关联性最强的circRNA进行环状性能鉴定。结果与对照组比较,40、80μg/ml CBNPs暴露72 h后16HBE细胞细胞活力明显下降,20、40、80μg/ml CBNPs暴露72 h后16HBE细胞LDH释放量明显上升(P<0.05)。与对照组比较,不同浓度CBNPs暴露72 h后16HBE细胞IL-6、IL-8 mRNA表达水平均增加,IL-6、IL-8蛋白水平均增高,TLR4、P-NF-κB、ASC、Caspase-1蛋白水平均明显上调,差异均有统计学意义(P<0.05)。与对照组比较,共有492个差异性表达的环状RNA(|log2 FC|>1),在验证出的5个差异表达(P<0.05)的环状RNA中,选择circ_002642作为后续环状RNA研究对象,80μg/ml CBNPs暴露72 h后16HB细胞的circ_002642明显高表达(P<0.05)。结论CBNPs可引起16HBE细胞炎症反应并诱导炎症反应环状RNA的差异性表达。

Objective To explore the toxic effect of carbon black nanoparticles on human bronchial epithelial cells,and identify the differentially expressed circular RNA based on the full transcriptome high-throughput sequencing,so as to provide evidence for the development of biomarkers exposed to carbon black nanoparticles and their application on epigenetic toxicology.Methods In June 2020,16 HBE cells were treated with carbon black nanoparticles at concentrations of 20,40 and 80μg/ml,and 16 HBE cells without any intervention were used as the control group.The cytotoxicity of carbon black nanoparticles was detected by CCK8 and LDH experiments.Real-time quantitative fluorescent PCR(qRT-PCR)and ELISA were used to detect the changes of interleukin-6(IL-6)and interleukin-8(IL-6,IL-8)mRNA and protein levels of carbon black nanoparticles with concentration gradient after 72 h exposure.Western blot analysis was conducted to detect the expression levels of toll-like receptor 4(TLR4),phosphorylated nuclear factor-κB(P-NF-κB),apoptosis-related speckled protein(ASC)and Caspase-1 associated with nuclear factor-κB.According to high-throughput sequencing results,differentially expressed Circrnas were screened and identified by qRT-PCR,and those with stable differentially expressed circrnas and the strongest association with the NF-κB pathway were selected for ring performance identification.Results After being exposed to carbon black nanoparticles for 72 h,the activity of 16HBE cells decreased significantly(P<0.05),and the release of lactate dehydrogenase increased significantly(P<0.05).Compared with control group,mRNA expression levels of IL-6 and IL-8,protein levels of IL-6 and IL-8 were increased,and protein levels of TLR4,p-NF-κB,ASC and Caspase-1 were significantly up-regulated in 16 HBE cells of different concentrations,with statistical significance(P<0.05).Compared with the control group,a total of 492 differentially expressed circular Rnas(|log2 FC|>1)were detected.Among the 5 differentially expressed(P<0.05)circular Rnas,circ_002642 was selected as the object of subsequent research on circular Rnas,affter 72 hours of exposure to 80μg/ml CBNPs,16HBE cells showed signlficantly higher expression of circ_002642(P<0.05).Conclusion Carbon black nanoparticles can induce differentially expressed circular RNAs associated with inflammatory response in human bronchial epithelial cells.