Abstract

Background:

Chronic arsenic exposure via drinking water is associated with an increased risk of developing cancer and noncancer chronic diseases. Pre-mRNAs are often subject to alternative splicing, generating mRNA isoforms encoding functionally distinct protein isoforms. The resulting imbalance in isoform species can result in pathogenic changes in critical signaling pathways. Alternative splicing as a mechanism of arsenic-induced toxicity and carcinogenicity is understudied.

Objective:

This study aimed to accurately profile differential alternative splicing events in human keratinocytes induced by chronic arsenic exposure that might play a role in carcinogenesis.

Methods:

Independent quadruplicate cultures of immortalized human keratinocytes (HaCaT) were maintained continuously for 28 wk with 0 or $100\text{\hspace{0.17em}nM}$ sodium arsenite. RNA-sequencing (RNA-Seq) was performed with poly(A) RNA isolated from cells harvested at 7, 19, and 28 wk with subsequent replicate multivariate analysis of transcript splicing (rMATS) analysis to detect and quantify differential alternative splicing events. Reverse transcriptase-polymerase chain reaction (RT-PCR) for selected alternative splicing events was performed to validate RNA-Seq predictions. Functional enrichment was performed by gene ontology (GO) analysis of the differential alternative splicing event data set at each time point.

Results:

At least 600 differential alternative splicing events were detected at each time point tested, comprising all the five main types of alternative splicing and occurring in both open reading frames (ORFs) and untranslated regions (UTRs). Based on functional relevance ELK4, SHC1, and XRRA1 were selected for validation of predicted alternative splicing events at 7 wk by RT-PCR. Densitometric analysis of RT-PCR data corroborated the rMATS predicted alternative splicing for all three events. Protein expression validation of the selected alternative splicing events was challenging given that very few isoform-specific antibodies are available. GO analysis demonstrated that the enriched terms in differential alternatively spliced mRNAs changed dynamically with the time of exposure. Notably, RNA metabolism and splicing regulation pathways were enriched at the 7-wk time point, when the greatest number of differentially alternatively spliced mRNAs are detected. Our preliminary proteomic analysis demonstrated that the expression of the canonical isoforms of the splice regulators DDX42, RMB25, and SRRM2 were induced upon chronic arsenic exposure, corroborating the splicing predictions.

Discussion:

These results using cultures of HaCaT cells suggest that arsenic exposure disrupted an alternative splice factor network and induced time-dependent genome-wide differential alternative splicing that likely contributed to the changing proteomic landscape in arsenic-induced carcinogenesis. However, significant challenges remain in corroborating alternative splicing data at the proteomic level. https://doi.org/10.1289/EHP9676

中文翻译：

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长期暴露于砷长达 28 周的 HaCaT 人角质形成细胞系中差异可变剪接的时间调制

摘要

背景：

通过饮用水长期接触砷会增加患癌症和非癌症慢性疾病的风险。Pre-mRNAs 通常受到选择性剪接，产生编码功能不同的蛋白质异构体的 mRNA 异构体。由此产生的异构体物种不平衡可导致关键信号通路的致病性变化。选择性剪接作为砷诱导的毒性和致癌性机制的研究不足。

客观的：

本研究旨在准确描述慢性砷暴露诱导的人类角质形成细胞中可能在致癌作用中起作用的差异选择性剪接事件。

方法：

永生化人角质形成细胞 (HaCaT) 的独立四份培养物连续维持 28 周，其中 0 或$100\text{\hspace{0.17em}纳米}$亚砷酸钠。使用从 7、19 和 28 周收获的细胞中分离的 poly(A) RNA 进行 RNA 测序 (RNA-Seq)，随后对转录本剪接 (rMATS) 分析进行重复多变量分析，以检测和量化差异选择性剪接事件。对选定的可变剪接事件进行逆转录酶-聚合酶链反应 (RT-PCR) 以验证 RNA-Seq 预测。通过在每个时间点对差异可变剪接事件数据集进行基因本体论 (GO) 分析来进行功能富集。

结果：

在每个测试的时间点检测到至少 600 个差异可变剪接事件，包括所有五种主要类型的可变剪接，并发生在开放阅读框 (ORF) 和非翻译区 (UTR) 中。基于功能相关性ELK4、SHC1和XRRA1被选择用于通过 RT-PCR 在 7 周时验证预测的可变剪接事件。RT-PCR 数据的光密度分析证实了 rMATS 预测的所有三个事件的可变剪接。鉴于可用的异构体特异性抗体非常少，对选定的可变剪接事件的蛋白质表达验证具有挑战性。GO分析表明，差异选择性剪接mRNA中的富集项随着暴露时间而动态变化。值得注意的是，RNA 代谢和剪接调节途径在 7 周时间点富集，此时检测到最大数量的差异剪接 mRNA。我们的初步蛋白质组学分析表明，剪接调节因子 DDX42、RMB25、

讨论：

这些使用 HaCaT 细胞培养物的结果表明，砷暴露破坏了选择性剪接因子网络并诱导了时间依赖性全基因组差异选择性剪接，这可能导致砷诱导的致癌作用中蛋白质组学景观的变化。然而，在蛋白质组水平上证实选择性剪接数据仍然存在重大挑战。https://doi.org/10.1289/EHP9676