Arsenic Induces Members of the mmu-miR-466-669 Cluster Which Reduces NeuroD1 Expression

Abstract

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 noncoding 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 4 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.

Long‐term arsenic exposure impairs differentiation in mouse embryonal stem cells

“…The expression of several early neural markers was also investigated. Previous studies focusing on acute arsenic exposure have shown a reduction in Ascl1 , a transcription factor needed for neural differentiation (Liu & Bain, 2014, 2018). Whereas Ascl1 mRNA expression was reduced at an acute 0.5‐μM arsenic exposure, it was not altered at the lower 0.1‐μM level.…”

“…PCR efficiency and linearity were determined using a standard curve generated by serial dilutions of five concentration points (10 −3 to 10 −7 ng DNA) of the gene of interest. The geometric mean of Gapdh and β2-microglobulin was used for normalization, as the expression of these reference genes does not vary due to arsenic exposure (Liu & Bain, 2018). Relative fold expression was compared with the Week 0 controls and quantified by the delta-delta Ct method (Schmittgen & Livak, 2008).…”

Intracerebral overexpression of miR-669c is protective in mouse ischemic stroke model by targeting MyD88 and inducing alternative microglial/macrophage activation

“…C2MC, also known as miR-297-669 cluster, is derived from intron 10 of the Polycomb group gene sex comb on the midleg with four MBT domains-2 (Sfmbt2) on mouse chromosome 2 [16]. MiRNA members of this cluster have been shown to be upregulated upon different harmful stimuli such as nutrient depletion conditions in Chinese hamster ovary (CHO) cells [17,18], acetaminophen-induced liver injury [19] and liver aging in mice [20], sodium arsenite exposure in P19 mouse embryonal carcinoma cells [21] and in the ischemic cortex 24 h after transient middle cerebral artery occlusion (tMCAo) in rats [22].…”