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Differentially Expressed mRNA Targets of Differentially Expressed miRNAs Predict Changes in the TP53 Axis and
Laila Al-Eryani1, Sabine Waigel2, Ashish Tyagi3
1Department of Pharmacology and Toxicology.
Abstract:
Arsenic is a widely distributed toxic natural element. Chronic arsenic ingestion causes several cancers, especially skin cancer. Arsenic-induced cancer mechanisms are not well defined, but several studies indicate that mutation is not the driving force and that microRNA expression changes play a role. Chronic low arsenite exposure malignantly transforms immortalized human keratinocytes (HaCaT), serving as a model for arsenic-induced skin carcinogenesis. Early changes in miRNA expression in HaCaT cells chronically exposed to arsenite will reveal early steps in transformation. HaCaT cells were maintained with 0/100 nM NaAsO2 for 3 and 7 weeks. Total RNA was purified. miRNA and mRNA expression was assayed using Affymetrix microarrays. Targets of differentially expressed miRNAs were collected from TargetScan 6.2, intersected with differentially expressed mRNAs using Partek Genomic Suite software, and mapped to their pathways using MetaCore software. MDM2, HMGB1 and TP53 mRNA, and protein levels were assayed by RT-qPCR and Western blot. Numerous miRNAs and mRNAs involved in carcinogenesis pathways in other systems were differentially expressed at 3 and 7 weeks. A TP53 regulatory network including MDM2 and HMGB1 was predicted by the miRNA and mRNA networks. Total TP53 and TP53-S15-phosphorylation were induced. However, TP53-K382-hypoacetylation suggested that the induced TP53 is inactive in arsenic exposed cells. Our data provide strong evidence that early changes in miRNAs and target mRNAs may contribute to arsenic-induced carcinogenesis.
Insights
Chronic arsenic exposure alters microRNA (miRNA) expression, contributing to skin cancer development. These early miRNA changes, not mutations, appear key in arsenic-induced carcinogenesis.
Area of Science:
- Environmental Toxicology
- Molecular Carcinogenesis
- Epigenetics
Background:
- Arsenic is a toxic element linked to various cancers, particularly skin cancer.
- The precise mechanisms of arsenic-induced carcinogenesis are unclear, but microRNA (miRNA) dysregulation is implicated over genetic mutation.
- Immortalized human keratinocytes (HaCaT) provide a model for studying arsenic's effects on skin cells.
Purpose of the Study:
- To investigate early changes in miRNA expression during chronic low-dose arsenite exposure in HaCaT cells.
- To identify molecular pathways affected by these early miRNA alterations in the context of skin carcinogenesis.
- To explore the role of the TP53 regulatory network in arsenic-induced transformation.
Main Methods:
- HaCaT cells were exposed to 100 nM sodium arsenite (NaAsO2) for 3 and 7 weeks.
- MicroRNA (miRNA) and messenger RNA (mRNA) expression profiling was performed using Affymetrix microarrays.
- Bioinformatic analyses (TargetScan, Partek, MetaCore) predicted miRNA targets and mapped them to biological pathways. TP53, MDM2, and HMGB1 levels were validated by RT-qPCR and Western blot.
Main Results:
- Numerous differentially expressed miRNAs and mRNAs involved in carcinogenesis pathways were identified at 3 and 7 weeks.
- A TP53 regulatory network, including MDM2 and HMGB1, was predicted based on miRNA and mRNA expression data.
- While total TP53 and its phosphorylation increased, hypoacetylation suggested impaired TP53 function in arsenite-exposed cells.
Conclusions:
- Early alterations in miRNA and target mRNA expression are significant contributors to arsenic-induced skin carcinogenesis.
- The identified TP53 regulatory network and impaired TP53 activity highlight potential mechanisms in arsenic toxicity.
- These findings underscore the role of epigenetic modifications, specifically miRNA changes, in the early stages of arsenic-related cancer development.
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