Differentially Expressed mRNA Targets of Differentially Expressed miRNAs Predict Changes in the TP53 Axis and Carcinogenesis-Related Pathways in Human Keratinocytes Chronically Exposed to Arsenic

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.

Figure 1.

Exposure and time-dependent differential expression of small RNAs and mRNAs detected by hybridization microarrays. A, Venn diagrams of differentially expressed small RNAs at both 3 and 7 weeks (p < .05). Unsupervised hierarchical clustering of the 19 small RNAs differentially expressed at both 3 and 7 weeks of exposure to arsenite. B, Arsenite exposure-dependent induced (red boxed) and suppressed (green boxed) small RNAs. C, Time-dependent differentially expressed miRNAs and the expression pattern is flipped by arsenite exposure compared with nonexposed cells, induced (red boxed) and suppressed (green boxed). D, Venn diagram of differentially expressed mRNAs at 3 and 7 weeks. E, Predicted mRNA targets of 6 differentially expressed miRNAs were found in TargetScan Human V6.2 database. F, Venn diagram of intersection of predicted mRNA targets of differentially expressed miRNAs and differentially expressed 323 mRNAs at both 3 and 7 weeks. Unsupervised hierarchical clustering of the 39 mRNAs differentially expressed at both 3 and 7 weeks of exposure to arsenite that are predicted targets of 6 miRNAs differentially expressed at both 3 and 7 weeks. G, Arsenite exposure-dependent induced (red boxed) and suppressed (green boxed) mRNAs. H, Time-dependent differentially expressed mRNAs and the expression pattern is flipped by arsenite exposure compared with nonexposed cells, induced (red boxed) and suppressed (green boxed). Analysis was done using Partek Genomic Suite.

Figure 2.

miRNA and target genes expression target TP53. Arsenite suppressed the expression miR-410, -548ac, and -548a-3p. MDM2 mRNA expression levels were induced at 3 weeks of arsenite exposure which might lead to the induction of MDM2 protein levels. HMGB1 mRNA expression levels were induced at 3 and 7 weeks of arsenite exposure which might lead to the induction in HMGB1 protein levels. Arsenite suppression of miR-410 and -548ac might induce MDM2 and HMGB1 at 3 weeks leading to the decrease in the levels of TP53.

Figure 3.

Arsenite exposure effects on HMGB1, MDM2, and TP53 expression. After 7 weeks chronic exposure (A) Western blot and quantitation showing no change in HMGB1. B, Western blot and quantitation showing increased expression of MDM2 isoform A (MDM2-A, 60 kDa), but not isoform MDM2-FL. C, Western blot and quantitation showing both TP53 and TP53 phosphorylated at serine 15 (TP53-S15P) were induced with arsenic exposure. D, Western blot and quantitation showing TP53 hypoacetylated at lysine 382 (TP53-K382Ac) in arsenite exposed cells. Proteins levels were normalized to β-actin levels and means ± SD are plotted. Student’s t-test was used for statistical analyses. *p-value ≤ .05. Samples from 4 independent cultures incubated without arsenite for 7 weeks (lanes 1–4) were compared with samples from 4 independent cultures incubated in parallel with 100 nM NaAsO₂ for 7 weeks (lanes 5–8). After probing for query proteins (HMGB1, MDM2, TP53), blots were stripped and probed for β-actin.