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. 2023 Apr 27;55(3):392–399. [Article in Chinese] doi: 10.19723/j.issn.1671-167X.2023.03.002

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

Jia-qi SHI 1, Ying MA 1, Yi ZHANG 1, Zhang-jian CHEN 1, Guang JIA 1,*
PMCID: PMC10258066  PMID: 37291912

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.

Keywords: Titanium dioxide nanoparticles, Hepatotoxicity, circRNA, Epigenomics

二氧化钛(titanium dioxide，TiO2)因其化学惰性、高折射率和低成本等特点，被广泛应用于光催化[1-2]、制药工业[3-4]和食品加工[5-6]等领域。纳米二氧化钛颗粒(titanium dioxide nanoparticles，TiO2 NPs)的粒径为1~100 nm，与微粒级尺寸相比具有更大的可催化表面积，因此纳米级颗粒会显示出更强的催化活性[7-8]。TiO2 NPs具有抗菌性及高折射率等特点，被普遍应用于食品加工等领域，因此食品级TiO2 NPs(在欧盟被称为E171，在美国被称为INS171)的安全性越来越受到关注。欧洲食品安全局认为E171作为食品添加剂是不安全的，2022年初，欧盟发布的委员会条例禁止将E171作为食品添加剂[9]，但中国仍允许其作为食品添加剂使用，因此对于TiO2 NPs经口暴露的安全性还需进行研究。

肝脏是人体内药物代谢的最主要器官，也是经口暴露于TiO2 NPs后的主要靶器官[10-12]，对于TiO2 NPs肝毒性的研究很有必要，故此本研究选择了一种常用于研究肝细胞毒性的细胞系，即人肝癌细胞(human hepatocellular carcinoma cells，HepG2)。除了细胞毒性和遗传毒性作用外，TiO2 NPs诱导的表观遗传变化也逐渐引起关注。表观遗传学是基因型和表型之间的重要纽带，在许多细胞生命进程的调节中起着关键作用，研究对象主要包括DNA甲基化、组蛋白修饰、非编码RNA、顺式调控元件以及染色质高级结构。其中，非编码RNA在许多细胞生命进程中起着重要作用，它们的失调与各种疾病的发病机制有关。环状核糖核酸(circular ribonucleic acid，circRNA)是一类特殊的非编码RNA分子，是共价闭环结构，缺乏游离的3′和5′末端，在物种间大量表达且高度保守[13]。circRNA可以调节转录和翻译[14]，参与蛋白质的螯合反应和易位[15]，促进蛋白质之间的相互作用[16]以及在蛋白质生物合成过程中起重要作用[17]。为了充分评估TiO2 NPs的毒性，分析TiO2 NPs的表观遗传作用至关重要。然而，目前有关纳米材料对circRNA影响的研究比较有限，尚缺乏关于circRNA在TiO2 NPs诱导毒性过程中的功能方面的研究。因此，本研究探讨了不同浓度TiO2 NPs处理HepG2细胞对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。

1. 材料与方法

1.1. 实验材料

HepG2细胞来自中国医学科学院基础医学研究所，北京协和医学院基础学院细胞资源中心。细胞培养液为Earle’s平衡盐溶液(EBSS)、L-谷氨酰胺的MEM培养基+10%(体积分数)胎牛血清+2%(质量分数)谷氨酰胺(200 mmol/L)+1%(体积分数)非必需氨基酸溶液(MEM无酚红)+1%(质量分数)青霉素-链霉素溶液。TiO2 NPs为球形锐钛矿，纯度在99.99%以上。详细的表征方法和结果参考本课题组前期发表的研究[18]。利用动态光散射(dynamic light scattering，DLS)测定无血清培养液中TiO2NPs的水合粒径。染毒液使用含EBSS、L-谷氨酰胺的MEM培养基溶解TiO2NPs至期望染毒浓度，充分超声分散并涡旋混匀。染毒时选取对数生长期的细胞，使用现配的相应浓度的TiO2NPs染毒液进行。

1.2. 细胞毒性检测

使用细胞计数试剂盒(cell counting kit-8，CCK8，购自Biotopped)测定TiO2 NPs的细胞毒性，暴露于0、1.56、3.13、6.25、12.5、25、50、100和200 mg/L TiO2 NPs 24 h和48 h后，将96孔板中的细胞与CCK8溶液共同孵育2 h，收集上清液，使用酶标仪检测，以600 nm为参比，检测450 nm处的光密度值。经过计算获得细胞活力结果，根据国家标准GB/T 16886.5—2017[19]，细胞活力下降至70%以下表明具有潜在细胞毒性，因此按照细胞活力大于70%的标准选取合适的染毒浓度和时间。

1.3. RNA测序

经过相应的染毒时间后先使用PBS缓冲液清洗残余染毒液，然后使用Trizol法提取RNA，凝胶电泳法对所提RNA进行质量检测，合格后纯化RNA并去除核糖体RNA(ribosomal RNA，rRNA)，构建测序样本文库，接着使用Illumina NovaSeq 6000测序仪进行测序，经过碱基识别及误差过滤，得到可以用于分析的原始测序片段。

1.4. circRNA表达量分析

运用CIRI软件进行circRNA预测，与circBase数据库进行比对，区分已知的circRNA和新的circRNA，利用perl脚本对预测的circRNA进行分类统计。使用SRPBM(spliced reads per billion mapping)[13]对序列进行归一化处理后，计算circRNA表达量。应用edgeR进行样本间差异circRNA分析，得出P值后采用错误发现率(false discovery rate，FDR)校正进行多重假设检验，同时根据SRPBM值计算差异倍数(fold change)。

根据SRPBM值计算出所有样品之间的相关性，然后通过主成分分析进行数据降维，通过正交偏最小二乘法判别分析(orthogonal projections to latent structures discriminant analysis，OPLS-DA)实现对样本类别的预测。差异基因筛选条件须同时满足P < 0.05以及差异倍数≥2。最后根据circRNA的位置信息，可以获得与circRNA所在基因组位置上对应的编码基因，对差异circRNA的匹配基因进行GO(Gene Ontology)和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。

1.5. Real-time RT-PCR验证

使用实时逆转录聚合酶链反应(real-time reverse transcription-polymerase chain reaction，real-time RT-PCR)验证测序分析结果，筛选显著改变的差异基因以及显著富集通路中的重要基因。

通过ICG网站(http://icg.big.ac.cn/index.php/Main_Page)得到HepG2细胞适用的内参基因GAPDH。目的基因通过查询PubMed网站(https://www.ncbi.nlm.nih.gov/gene/)的“Primer-BLAST”设计相应的引物序列。目的基因和内参基因的引物序列见表 1。

表 1.

目的基因和内参基因的引物序列

Primer sequences of the target gene and the internal reference gene

Target gene Upstream primers (5′-3′) Downstream primers (3′-5′)
circRNA, circular ribonucleic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
circRNA.6730 CTGAACCTTGCTCCGAGAGG GAACTCAGAAACCGCAGGGA
circRNA.3650 AGGGCTCCGCTTTATTTGCT CAGATTCCTAACTGTCTGGAGGG
circRNA.10113 AGACTGAAGGAGCAGTTGCC TCCTCGTGCAAGGATTTCCC
circRNA.4321 CCCAGGGAACCAATCTGTCC CACAGCAAGGCCTGGAGTTA
GAPDH TGCAACGGCGGAAGAAAA ACGAGGCTTTCAATGTTGCC
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1.6. 数据分析

统计分析由R4.1.0软件完成，利用simca 14.1软件作图，每个样本均设置三次重复平行样，使用Shapiro-Wilk正态性检验和Bartlett方差齐性检验，连续变量数据均以均值±标准差表示，所有检验均为双侧检验，P < 0.05为差异有统计学意义。

2. 结果

2.1. TiO2 NPs表征

表征结果显示，本研究使用的TiO2 NPs为球形锐钛矿，等效直径为(25.12±5.64) nm，在无血清培养基(1 g/L TiO2 NPs)体系中的水合粒径为(323.50±85.44) nm，Zeta电位为(-21.00±0.72) mV，纯度>99.99%，呈现团聚状态。

2.2. 细胞毒性

染毒24 h和48 h后，随着TiO2 NPs浓度的增加，细胞活力逐渐下降(图 1)。染毒48 h时，200 mg/L组的细胞活力(65.25%)较对照组(0 mg/L TiO2 NPs)显著下降，但由于200 mg/L组的细胞活力过低，我们最终选择了100 mg/L TiO2 NPs (细胞活力74.16%)作为染毒组，染毒时间为48 h。

图 1.

TiO2 NPs的细胞毒性

Cytotoxicity of TiO2 NPs

*P < 0.05, compared with the 0 mg/L TiO2 NPs group.

图 1

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2.3. circRNA的定量和特征

数据预处理后获得11 478个circRNA，主要分为外显子、基因区间和内含子三类，分别有9 591、405和1 482个，其中染毒组和对照组占最大比例的均为外显子(图 2A)。主成分分析显示染毒组与对照组略分离(图 2B)，OPLS-DA分析显示染毒组与对照组分离效果好，表示circRNA特征表达具有差异性(图 2C)。为防止过拟合，采用置换检验进行验证(n=200)，100 mg/L TiO2 NPs染毒组与对照组相比，RX2=0.597、RY2=1、Q2=1，说明获得的数据可靠，模型对差异的识别能力强、预测能力好。

图 2.

TiO2 NPs染毒后的circRNA表达量分析

Analysis of circRNA expression after TiO2 NPs exposure

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 2

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2.4. circRNA差异表达量分析

绘制相关性分析散点图发现，染毒组与对照组的表达呈极弱正相关关系(图 3A)。差异表达的circRNA火山图结果见图 3B，按照P < 0.05、circRNA差异表达倍数≥2的筛选标准进行筛选，最终发现与对照组相比，染毒组共有89个差异circRNA，其中外显子占多数，有77个。在所有差异表达的circRNA中，59个显著上调，如circRNA.9204、circRNA.10311和circRNA.7990等；30个显著下调，如circRNA.4650、circRNA.4321和circRNA.10113等。TiO2 NPs染毒组与对照组之间差异circRNA的热图如图 3C所示，直观地表明了染毒组和对照组对circRNA的影响不同。

图 3.

TiO2 NPs染毒后的circRNA差异表达分析

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

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 3

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2.5. 差异circRNA富集通路分析

基于差异表达circRNA的靶基因进行GO富集分析，结果显示生物过程分类中基因富集程度排前三位的是细胞生命进程、代谢过程和单分子过程；细胞成分分类中排前三位的是细胞组分、细胞和细胞器；分子功能分类中排前三位的是结合、催化活性和核酸结合转录因子活性(图 4A)。

图 4.

差异circRNA的GO和KEGG富集分析

GO and KEGG enrichment analysis of differential circRNA

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 4

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基于差异表达circRNA的靶基因进行KEGG富集分析，结果显示组织系统分类中基因富集程度排前三位的通路均与内分泌系统相关；细胞生命进程分类中排前三位的通路为细胞集群、运输和分解代谢；环境信息处理分类中排前三位的通路均与信号转导相关；遗传信息处理分类中排前三位的通路与复制、修复以及折叠、分类和降解相关；人体疾病分类中排前三位的通路均为癌症；代谢分类中排前三位的通路为脂质代谢、氨基酸代谢和能量代谢。KEGG富集通路分析结果显示，富集程度排前三位的通路为脂肪酸降解、范可尼贫血(Fanconi anemia)通路以及脂肪酸代谢(图 4B)。富集在脂肪酸降解和脂肪酸代谢通路上的差异circRNA均为circRNA.6730和circRNA.6580，富集在范可尼贫血通路上的差异circRNA为circRNA.3650和circRNA.3971。

2.6. Real-time RT-PCR验证

根据circRNA差异表达分析筛选出差异最显著的circRNA.10113和circRNA.4321，从显著富集的脂肪酸降解及范可尼贫血通路中选择差异表达的circRNA.6730和circRNA.3650，最终选出4个circRNA进行验证实验。如图 5的real-time RT-PCR结果显示，100 mg/L TiO2 NPs染毒48 h后，circRNA.6730、circRNA.3650和circRNA.4321的相对表达量与对照组相比均显著降低，与测序结果一致。

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

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

* 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.

图 5

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3. 讨论

本研究的CCK8细胞毒性检测结果表明，TiO2 NPs暴露会导致HepG2细胞活力下降，产生细胞毒性。很多研究已经证实，肝毒性是TiO2NPs经口暴露的主要靶器官之一[12, 20]。也有研究发现肝脏是对TiO2 NPs诱导的氧化应激最敏感的组织[21]，因此对于TiO2 NPs诱导的肝毒性研究很有必要。在纳米材料领域已经有很多研究利用HepG2细胞系来检测潜在肝毒性，具有一定的可靠性和可信度。Kitchin等[22]使用了5种不同浓度的TiO2 NPs对HepG2细胞进行染毒，均发现了不同程度的细胞毒性，与本研究结果一致。

以往研究显示，表观遗传修饰是可遗传的，并且可以通过保留表观遗传记忆在初始刺激暴露后持续存在[23]，而纳米材料会诱导DNA甲基化模式[24]、组蛋白修饰[25]或者microRNA表达改变[26]，从而影响表观遗传变化，这表明纳米材料可能对人类健康存在潜在的长期影响。本研究发现TiO2 NPs可诱导肝细胞circRNA表达谱改变，并筛选出了89个差异circRNA，表明TiO2 NPs可以引起肝细胞表观遗传学改变。表观遗传修饰是调控基因型和表型的重要环节，它们的调控和失调往往导致疾病的发生和长期的负面影响。表观遗传学已开始逐步应用于纳米材料的毒性研究，有研究发现低浓度的TiO2 NPs可以改变负责表观遗传修饰的酶[27]，但该浓度远低于亚致死水平，因此，表观遗传学研究对于全面评估TiO2 NPs暴露的潜在风险至关重要，但目前尚缺乏TiO2 NPs与circRNA的关联研究。Li等[28]用由甘草酸和锌离子组成的金属纳米载体处理原代肝细胞，发现过度表达的circRNA_0001805可以通过抑制脂质代谢紊乱和炎症减轻非酒精性脂肪肝的进展。本研究通过real-time RT-PCR验证发现，TiO2 NPs染毒会引起circRNA.4321显著下调，其所在基因组位置上对应的编码基因为ZNF562，可参与转录的调节及DNA模板化[29]，因此circRNA.4321显著下调可能影响DNA结合转录因子活性。

本研究的通路富集分析发现，TiO2 NPs暴露可能干扰脂肪酸降解、范可尼贫血通路以及脂肪酸代谢等通路，最终导致肝细胞毒性。一些体内研究也同样发现TiO2 NPs暴露会诱导脂质代谢的变化。Federici等[30]将虹鳟鱼暴露于TiO2 NPs后，发现其肝细胞表现出轻微的脂肪变化和脂质化。但Volkovova等[31]对Wistar大鼠静脉注射0.592 mg/kg TiO2 NPs后并未发现其肝组织中的胆固醇和甘油三酯浓度产生变化，需要进一步研究脂质代谢在TiO2 NPs诱导的肝细胞毒性中的作用，为解释和开展相关体内试验提供线索。脂肪酸降解通路上的circRNA.6730(HADHB基因)显著下调，该基因编码的蛋白可催化线粒体β-氧化途径[32]，该途径是组织中的主要能量产生过程，因此circRNA.6730显著下调可能阻碍线粒体产生能量；范可尼贫血通路上的circRNA.3650(BRIP1基因)显著下调，范可尼贫血通路已被确定为一种DNA修复途径，可清除阻碍DNA复制和转录的屏障[33]。BRIP1的表达与许多肿瘤中的DNA甲基化水平呈负相关，并且与细胞凋亡、细胞周期及DNA损伤反应有关[34]。本研究发现TiO2 NPs暴露导致BRIP1表达水平显著下降，这可能阻碍DNA修复进而造成遗传毒性。

总之，本研究主要关注暴露于TiO2 NPs后HepG2细胞中circRNA表达谱的改变及其在肝细胞毒性机制中的潜在作用，结果表明，暴露于TiO2 NPs可以诱导以circRNA.6730、circRNA.3650和circRNA.4321为代表的一系列差异circRNA变化，提示表观遗传学可能在TiO2 NPs诱导肝细胞毒性的机制中发挥重要作用。

Funding Statement

国家自然科学基金(81703257)和科技部国家重点研发计划(2017YFC1600200)

Supported by the National Natural Science Foundation of China (81703257) and the National Key R & D Program of the Ministry of Science and Technology of China (2017YFC1600200)

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