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Journal of Peking University (Health Sciences) ›› 2023, Vol. 55 ›› Issue (3): 392-399. doi: 10.19723/j.issn.1671-167X.2023.03.002

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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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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

CLC Number: 

  • R114

Table 1

Primer sequences of the target gene and the internal reference gene"

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

Figure 1

Cytotoxicity of TiO2 NPs"

Figure 2

Analysis of circRNA expression after TiO2 NPs exposure"

Figure 3

The circRNA differential expression analysis after TiO2 NPs exposure of TiO2 NPs"

Figure 4

GO and KEGG enrichment analysis of differential circRNA"

Figure 5

Real-time RT-PCR verification results of four differentially expressed genes after TiO2 NPs exposure for 48 h"

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