Chronic arsenic exposure can result in adverse development effects including decreased intellectual function, reduced birth weight, and altered locomotor activity. Previous in vitro studies have shown that arsenic inhibits stem cell differentiation. MicroRNAs (miRNAs) are small non-coding RNAs that regulate multiple cellular processes including embryonic development and cell differentiation. The purpose of this study was to examine whether altered miRNA expression was a mechanism by which arsenic inhibited cellular differentiation. The pluripotent P19 mouse embryonal carcinoma cells were exposed to 0 or 0.5 μM sodium arsenite for 9 days during cell differentiation, and changes in miRNA expression was analyzed using microarrays. We found that the expression of several miRNAs important in cellular differentiation, such as miR-9 and miR-199 were decreased by 1.9- and 1.6-fold respectively following arsenic exposure, while miR-92a, miR-291a, and miR-709 were increased by 3-, 3.7- and 1.6-fold, respectively. The members of the miR-466-669 cluster and its host gene, Scm-Like with four Mbt domains 2 (Sfmbt2), were significantly induced by arsenic from 1.5- to 4-fold in a time-dependent manner. Multiple miRNA target prediction programs revealed that several neurogenic transcription factors appear to be targets of the cluster. When consensus anti-miRNAs targeting the miR-466-669 cluster were transfected into P19 cells, arsenic-exposed cells were able to more effectively differentiate. The consensus anti-miRNAs appeared to rescue the inhibitory effects of arsenic on cell differentiation due to an increased expression of NeuroD1. Taken together, we conclude that arsenic induces the miR-466-669 cluster, and that this induction acts to inhibit cellular differentiation in part due to a repression of NeuroD1.

Expression changes in differentiating P19 cells after arsenc exposureP19 cells were aggregated into embryoid bodies and then allowed to differentiate for a total of 9 days. Cells were continuously exposed to 0 or 0.5uM arsenite during this process, along with 1% dimethylsulfoxide (DMSO) to induce differentiation. Cells were collected on day 9. Data file is microRNA expression using LC Science's murine miRNA microarray (Geo platform GPL 21312).Array data for Dryad.csv