纳米二氧化钛颗粒对人肝癌细胞HepG2中circRNA表达谱的影响

Effects of titanium dioxide nanoparticles on circRNA expression profiles in human hepatocellular carcinoma cells HepG2

目的:通过体外细胞实验探讨纳米二氧化钛颗粒(titanium dioxide nanoparticles,TiO_(2)NPs)对人肝癌细胞(human hepatocellular carcinoma cells,HepG2)中环状核糖核酸(circular ribonucleic acid,circRNA)表达谱的影响,并通过生物信息学分析TiO_(2)NPs肝细胞毒性的潜在机制。方法:分别从粒径、形状、团聚状态等方面对TiO_(2)NPs进行表征,在暴露于0、1.56、3.13、6.25、12.5、25、50、100和200 mg/L TiO_(2)NPs 24 h或48 h后,利用细胞计数试剂盒(cell counting kit-8,CCK8)检测TiO_(2)NPs对HepG2的细胞毒性。以0 mg/L(对照组)和100 mg/L(染毒组)的TiO_(2)NPs处理HepG2细胞48 h后,收集细胞样本,提取RNA并进行测序。筛选出对照组和TiO_(2)NPs染毒组之间的差异circRNA,通过多变量统计分析差异circRNA靶基因的富集通路。根据测序结果,筛选出显著改变的基因以及显著富集通路中的重要基因,对HepG2细胞进行实时逆转录聚合酶链反应(real-time reverse transcription-polymerase chain reaction,real-time RT-PCR)验证。结果:TiO_(2)NPs为球形锐钛矿,在无血清培养基中的水合粒径为(323.50±85.44)nm,Zeta电位为(-21.00±0.72)mV。CCK8细胞毒性检测结果发现,随着TiO_(2)NPs浓度的增加,细胞活力逐渐下降。RNA测序共发现11478个circRNA,与对照组相比,TiO_(2)NPs染毒组(100 mg/L)中共有89个差异circRNA,其中59个上调,30个下调。根据日本京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析,差异circRNA的靶向基因主要富集在脂肪酸降解、范可尼贫血(Fanconi anemia)通路以及脂肪酸代谢等通路上。Real-time RT-PCR验证结果显示,代表性差异circRNA(包括circRNA.6730、circRNA.3650和circRNA.4321)的相对表达量在TiO_(2)NPs染毒组和对照组之间差异有统计学意义,与测序结果一致。结论:TiO_(2)NPs可诱导circRNA表达谱发生改变,提示表观遗传学可能在肝细胞毒性机制中发挥重要作用。

Objective:To investigate the effect of titanium dioxide nanoparticles(TiO_(2) NPs)on the expression profile of circular ribonucleic acid(circRNA)in human hepatocytes through in vitro cell experiments,and to attempt to understand the potential mechanism of hepatotoxicity through bioinformatics analysis.Methods:TiO_(2) NPs were characterized from the aspects of particle size,shape and agglomeration state.The cell counting kit-8(CCK8)was used to detect the cytotoxicity of TiO_(2) NPs against human hepatocellular carcinoma cells(HepG2)after exposure to 0,1.56,3.13,6.25,12.5,25,50,100,and 200 mg/L TiO_(2) NPs for 24 h or 48 h.The cells were treated at doses of 0 mg/L TiO_(2) NPs(control group)and 100 mg/L TiO_(2) NPs(treatment group),and collected after exposure for 48 h,and then RNA from the extracted cell samples was collected and sequenced.The differential circRNAs between the control and the TiO_(2) NPs treatment groups were screened,and then the enrichment pathway of the differential circRNA target gene was analyzed by multivariate statistics.According to the sequencing results,significantly altered genes and important genes in the significant enrichment pathways were screened,and real-time reverse transcription-polymerase chain reaction(real-time RT-PCR)was performed to verify the results.Results:TiO_(2) NPs were spherical anatase with a hydrated particle size of(323.50±85.44)nm and a Zeta potential of(-21.00±0.72)mV in a serum-free medium.The results of the CCK8 cytotoxicity assay showed that with the increase of TiO_(2) NPs concentration,cell viability gradually decreased.A total of 11478 circRNAs were found by RNA sequencing.Compared with the control groups,TiO_(2) NPs treatment groups(100 mg/L)had a total of 89 differential circRNAs,of which 59 were up-regulated and 30 were down-regulated.Analysis of the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway showed that the targeted genes of differential circRNAs were mainly enriched in fatty acid degradation,Fanconi anemia pathway,and fatty acid metabolism.The expression levels of circRNA.6730,circRNA.3650 and circRNA.4321 were significantly different between the TiO_(2) NPs treatment group and the control group,which were consistent with the sequencing results.Conclusion:TiO_(2) NPs can induce changes in circRNA expression profile,and epigenetics may play an important role in the mechanism of hepatotoxicity.