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北京大学学报（医学版） ›› 2023, Vol. 55 ›› Issue (3): 392-399. doi: 10.19723/j.issn.1671-167X.2023.03.002

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纳米二氧化钛颗粒对人肝癌细胞HepG2中circRNA表达谱的影响

史佳琪,马莺,张奕,陈章健,贾光*()   

  1. 北京大学公共卫生学院劳动卫生与环境卫生学系，食品安全毒理学研究与评价北京市重点实验室，北京 100191
  • 收稿日期:2023-02-06 出版日期:2023-06-18 发布日期:2023-06-12
  • 通讯作者: 贾光 E-mail:jiaguangjia@bjmu.edu.cn
  • 基金资助:
    国家自然科学基金(81703257);科技部国家重点研发计划(2017YFC1600200)

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

Jia-qi SHI,Ying MA,Yi ZHANG,Zhang-jian CHEN,Guang JIA*()   

  1. Department of Occupational and Environmental Health Sciences, Peking University School of Public Health; Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Beijing 100191, China
  • Received:2023-02-06 Online:2023-06-18 Published:2023-06-12
  • Contact: Guang JIA E-mail:jiaguangjia@bjmu.edu.cn
  • Supported by:
    the National Natural Science Foundation of China(81703257);the National Key R & D Program of the Ministry of Science and Technology of China(2017YFC1600200)

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摘要：

目的: 通过体外细胞实验探讨纳米二氧化钛颗粒(titanium dioxide nanoparticles，TiO2 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TiO2 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TiO2 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 TiO2 NPs 24 h或48 h后，利用细胞计数试剂盒(cell counting kit-8，CCK8)检测TiO2 NPs对HepG2的细胞毒性。以0 mg/L(对照组)和100 mg/L(染毒组)的TiO2 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TiO2 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: TiO2 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TiO2 NPs浓度的增加，细胞活力逐渐下降。RNA测序共发现11 478个circRNA，与对照组相比，TiO2 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TiO2 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: TiO2 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。

关键词: 二氧化钛纳米颗粒, 肝细胞毒性, 环状RNA, 表观基因组学

Abstract:

Objective: To investigate the effect of titanium dioxide nanoparticles (TiO2 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: TiO2 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 TiO2 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 TiO2 NPs for 24 h or 48 h. The cells were treated at doses of 0 mg/L TiO2 NPs (control group) and 100 mg/L TiO2 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 TiO2 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: TiO2 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 TiO2 NPs concentration, cell viability gradually decreased. A total of 11 478 circRNAs were found by RNA sequencing. Compared with the control groups, TiO2 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 TiO2 NPs treatment group and the control group, which were consistent with the sequencing results. Conclusion: TiO2 NPs can induce changes in circRNA expression profile, and epigenetics may play an important role in the mechanism of hepatotoxicity.

Key words: Titanium dioxide nanoparticles, Hepatotoxicity, circRNA, Epigenomics

中图分类号: 

  • R114

表1

目的基因和内参基因的引物序列"

Target gene Upstream primers (5′-3′) Downstream primers (3′-5′)
circRNA.6730 CTGAACCTTGCTCCGAGAGG GAACTCAGAAACCGCAGGGA
circRNA.3650 AGGGCTCCGCTTTATTTGCT CAGATTCCTAACTGTCTGGAGGG
circRNA.10113 AGACTGAAGGAGCAGTTGCC TCCTCGTGCAAGGATTTCCC
circRNA.4321 CCCAGGGAACCAATCTGTCC CACAGCAAGGCCTGGAGTTA
GAPDH TGCAACGGCGGAAGAAAA ACGAGGCTTTCAATGTTGCC

图1

TiO2 NPs的细胞毒性 *P < 0.05, compared with the 0 mg/L TiO2 NPs group."

图2

TiO2 NPs染毒后的circRNA表达量分析 The perl script was used to classify and plot the predicted circRNA to visualize the composition of circRNA (A). Principal component analysis (B) and OPLS-DA (C) based on the expression of credible circRNA were performed to compare differences between the control group and the treatment group. C1, 0 mg/L TiO2 NPs-control group 1; C2, 0 mg/L TiO2 NPs-control group 2; C3, 0 mg/L TiO2 NPs-control group 3; T1, 100 mg/L TiO2 NPs-treatment group 1; T2, 100 mg/L TiO2 NPs-treatment group 2; T3, 100 mg/L TiO2 NPs-treatment group 3. PC1, the first principal component; PC2, the second principal component; PCo1, the first orthogonal principal component. TiO2 NPs, titanium dioxide nanoparticles; circRNA, circular ribonucleic acid; OPLS-DA, orthogonal projections to latent structures discriminant analysis."

图3

TiO2 NPs染毒后的circRNA差异表达分析 The scatter plot of correlation analysis between the treatment group and the control group intuitively showed the extremely weak positive correlation (A). The volcano plot of differentially expressed genes in the treatment group showed the number of up-regulated and down-regulated genes (B). Cluster analysis heatmaps of the treatment and control groups showed differences in their characteristics (C). SRPBM, spliced reads per billion mapping. Other abbreviations as in Figure 2."

图4

差异circRNA的GO和KEGG富集分析 A, the results of the GO (Gene Ontology) enrichment analysis, with red representing biological processes, green representing cellular components, and blue representing molecular functions. B, the results of the KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis, and the dot size indicated the number of target genes enriched in this pathway, and the larger the shape, the greater the number. The color indicated the rank of the corrected P value, the greener the color, the larger the P value, and the redder the color, the smaller the P value. TGF, transforming growth factor; MAPK, mitogen activated protein kinase; Other abbreviations as in Figure 2."

图5

TiO2 NPs染毒48 h后4个差异表达基因的real-time RT-PCR验证结果 * P < 0.05, compared with the control group. TiO2 NPs, titanium dioxide nanoparticles; circRNA, circular ribonucleic acid; real-time RT-PCR, real-time reverse transcription-polymerase chain reaction."

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ISSN 1671-167X
CN 11-4691/R
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