Temporal Modulation of Differential Alternative Splicing in HaCaT Human Keratinocyte Cell Line Chronically Exposed to Arsenic for up to 28 Wk.
Temporal Modulation of Differential Alternative Splicing in HaCaT Human Keratinocyte Cell Line Chronically Exposed to Arsenic for up to 28 Wk.
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DOI：
10.1289/ehp9676
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发表时间：
2022-01
影响因子：
10.4
通讯作者：
States JC
中科院分区：
文献类型：
--
作者：
Ferragut Cardoso AP;Banerjee M;Al-Eryani L;Sayed M;Wilkey DW;Merchant ML;Park JW;States JC
通过饮用水长期接触砷与患癌症和非癌症慢性疾病的风险增加有关。前mRNA通常会发生选择性剪接，产生编码功能不同的蛋白质亚型的mRNA亚型。由此导致的异构体物种失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毒性和致癌性的机制尚未得到充分研究。本研究旨在准确描述慢性砷暴露诱导的人角化细胞中可能在致癌过程中起作用的不同选择性剪接事件。用0或亚砷酸钠连续培养永生化人角质形成细胞（HaCaT） 28周。RNA测序（RNA- seq）使用从7、19和28周收获的细胞中分离的poly(A) RNA进行，随后进行转录剪接的重复多变量分析（rMATS）分析，以检测和量化不同的可选剪接事件。逆转录聚合酶链反应（RT-PCR）对选择的备选剪接事件进行验证RNA-Seq预测。通过基因本体（GO）分析每个时间点的差异选择性剪接事件数据集进行功能富集。在每个时间点检测到至少600个不同的选择性剪接事件，包括所有五种主要类型的选择性剪接，发生在开放阅读框（orf）和非翻译区（utr）。基于功能相关性，选择ELK4、SHC1和XRRA1，通过RT-PCR验证7周时预测的可选剪接事件。RT-PCR数据的密度分析证实了rMATS预测了所有三个事件的选择性剪接。考虑到很少有同种异型特异性抗体可用，所选择的选择性剪接事件的蛋白表达验证具有挑战性。氧化石墨烯分析表明，差异选择性剪接mrna中的富集项随着暴露时间的变化而动态变化。值得注意的是，RNA代谢和剪接调节途径在7周时间点富集，此时检测到的差异选择性剪接mrna数量最多。我们初步的蛋白质组学分析表明，剪接调节因子DDX42、RMB25和SRRM2的典型亚型的表达在慢性砷暴露下被诱导，证实了剪接的预测。这些使用HaCaT细胞培养的结果表明，砷暴露破坏了替代剪接因子网络，并诱导了时间依赖性全基因组差异替代剪接，这可能导致砷诱导致癌过程中蛋白质组学景观的变化。然而，在蛋白质组学水平上确认替代剪接数据仍然存在重大挑战。https://doi.org/10.1289/EHP9676
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. 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. Independent quadruplicate cultures of immortalized human keratinocytes (HaCaT) were maintained continuously for 28 wk with 0 or 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. 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. 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
异常选择性剪接与甲状腺乳头状癌发生及预后相关的综合分析
DOI：
10.18632/aging.203608
发表时间：
2021-10-08
期刊：
Aging
影响因子：
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作者：
Zheng X;Feng L;Yin Y;Yu C;He X;Zhu J;Zhang M;Yu J;Xu M
通讯作者：
Xu M
影响因子：
3.4
作者：
Berglund, Susanne R.;Santana, Alison R.;Li, Dan;Rice, Robert H.;Rocke, David M.;Goldberg, Zelanna
通讯作者：
Goldberg, Zelanna