Temporal Modulation of Differential Alternative Splicing in HaCaT Human Keratinocyte Cell Line Chronically Exposed to Arsenic for up to 28 Wk

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 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 lands...

Comparative proteoinformatics revealed the essentials of SDS impact on HaCaT keratinocytes

“…GO analysis suggested that “slicing, via spliceosome” was slightly activated due to SDS exposure. Earlier, it was shown that alternative splicing induction may contribute to the changing proteomic landscape as at carcinogenesis, for example, in HaCaT cells after arsenic exposure 33 . In contrast, several metabolic pathways, such as negative regulation of apoptotic process and epidermis development, were downregulated after treatment of HaCaT cells with SDS.…”

Targeting alternative splicing in cancer immunotherapy

“…In addition to algorithm limitations, sequencing technology also encounters obstacles in identifying AS events. Currently, most studies identify and quantify splicing isoforms starting from bulk short-read RNA-seq data ( Cieślik and Chinnaiyan, 2018 ; Ferragut Cardoso et al, 2022 ; Toffali et al, 2023 ). These studies typically map the short-read RNA sequences to a reference genome using software such as MISO ( Katz et al, 2010 ) or rMATS ( Shen et al, 2014 ), or assemble de novo using tools including StringTie ( Pertea et al, 2015 ) or Trinity ( Grabherr et al, 2011 ).…”

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“…Cell lysis and immunoblotting were performed as previously described ( Ferragut Cardoso et al, 2022 ). Briefly, cells were lysed using lysis buffer (10 mM Tris – HCl pH 7.4, 1 mM Na 2 − EDTA, 0.1 % SDS, 1 mM Na-Vanadate, 1 mM PMSF and 1 X protease inhibitor cocktail (Complete, Sigma-Aldrich).…”