Altered Mir Expression in Arsenic-Exposed Hvm1 Cells During Differentiation

Neurological abnormalities are included in the main effects of chronic lower level arsenic exposure in humans due to the consumption of contaminated drinking water. Recently, epigenetic mechanisms may explain the effects of xenobiotics. Arsenic exposure alters DNA methyltransferase (DNMT) enzyme expression and activity. It was interesting to determine if arsenic can modify other epigenetic mediators. Recently, the microRNA has been shown to regulatory effects at several molecular targets and regulate physiological processes. We performed a study to determine whether hVM1 cells exposed to arsenic during differentiation have altered expression levels of miR-125b,-148a and -132, and if these changes are associated with gene expression changes in nestin, CREB, BNDF and DNMT3b using real time RT-PCR. Statistically significant changes were observed in miR-125b and miR-148a expression. Although hVM1 cells exposed to arsenic showed a trend to downregulate miR-132, this was not significant. Nestin, an important marker of diminished differentiation, was negatively correlated with miR-125b and showed a trend for increased expression that may have a physiological interpretation. In conclusion, these results show that arsenic exposure could lead changes in the expression of key microRNA and therefore could affect neural differentiation in hVM1 cells.


Introduction
Chronic arsenic exposure is a global environmental health problem that affects millions of people worldwide. There are several regions in which drinking water exceeds the Environmental Protection Agency (EPA) limit of 10 ppb (10μg/L) and the local limit of arsenic (NOM-127 25μg/L). Several long-term health effects of low dose arsenic exposure in humans have been reported; the most important of these are the association with an increased risk of epithelial cancers of the skin, bladder, lung, liver, kidney and prostate, cardiovascular diseases, genotoxic and immunotoxic effects and, interestingly, neurological abnormalities [1][2][3][4].
Furthermore, arsenic exposure through drinking water induces general embryotoxicity and damages embryonic neurogenesis [5][6][7] During embryogenesis, arsenite exposure might negatively affect the proliferation, survival and differentiation of embryonic neural stem cells [8]. The mechanisms that have been attributed to arsenic involve a wide range of effects; although debate continues, there is evidence to support the disruption of signaling cascades [9,10], oxidative stress [11] and epigenetic changes [12,13]. Recently, the field of epigenetics has opened up, allowing researchers to examine molecular mechanisms in response to gene-environment interactions and to elucidate how toxic compounds have broad effects. The wide effects associated to arsenic exposure suggest that arsenic is able to modify the responses through epigenetic mechanism. Epigenetic mechanisms allow cells to rapidly alter their long-term transcriptional activity, thereby permitting coordinated changes in gene expression without permanently altering the sequence of DNA. DNA methylation is one of the most frequent epigenetic alterations described following exposure to arsenic. It has been reported that arsenic is able to alter the activity of DNA methyltransferase (DNMT). DNMTs catalyze methyl group transfer from S-adenosylmethionine (SAM) into the C5´position of cytosine at CpG dinucleotides to produce 5-methylcytosine on promoter regions of genes; this silences gene expression [14,15].
The inhibition of DNMT activity by arsenic exposure appears to be a mechanism related to whole-genome and gene-specific DNA demethylation. However, in contrast, more recent reports have shown that localized DNA hypermethylation due to arsenic exposure is challenging to explain. [16] showed that localized DNA hypomethylation and hypermethylation occur following chronic arsenic exposure in Wistar rats. They observed a significant increase in the non-methylated form of the PP1 gene promoter, both in the cortex and hippocampus. At the same time, they observed an increase in the methylated form of the RELN gene promoter.
Therefore, arsenic exposure may alter the methylation pattern of neural plasticity genes, but the mechanisms involved may be additional to or complemented by DNMT activity and methyl group consumption.
On the other hand, epigenetics has been recently been extended to microRNA (miR), which are small non-coding RNA molecules of about 21-23 nucleotides that function is posttranscriptional negative regulation of gene expression [17]. They have been implicated in the control of every cellular physiological process, including neural differentiation and maintenance of the neural phenotype [18,19] as well as in brain development and function [20][21][22]. miR-125b is highly expressed in the central nervous system, and is upregulated during neural differentiation in embryonic stem cells [23,25] demonstrated that miR-125b suppresses the expression of nestin (a marker of multipotent neural stem cells) by binding to the 3´-UTR of its mRNA, such that the expression of this target is downregulated when miR-125b is upregulated. Another miR investigated in the present study is miR-132, which plays role in neural development and neural activity and is able to negatively modulate neural differentiation by regulating various genes [25,26]. In addition, miR-148a is an important regulator of DNMT3b expression [27], which is involved in the establishment of new methylation patterns during development and is most important during neural differentiation [28]. Evidence that chemical carcinogen exposure is capable of altering miRNA expression continues to emerge [29,30]. However, if miR-125b, miR-132 and 148a expression can be altered by arsenic exposure, the implications for neural differentiation remain unknown. A new tool to evaluate the effect of arsenic exposure on neural differentiation is a stable cell line of human neural stem cells derived from the ventral mesencephalon (hVM1). hVM1 cells were isolated from a 10-weekold aborted human fetus, and immortalization was carried out by infection with a retroviral vector coding for v-myc (LTR-vmyc-SV40-Neo-LTR). This polyclonal cell line has the capacity for selfrenewal and is able to differentiate into neurons, astrocytes and oligodendrocytes [31][32][33]. In this study, we investigated the effect of arsenic exposure on miR-125b, miR-132 and miR-148a expression and additionally some target genes related to neural differentiation (nestin, CREB, BNDF and DNMT3b) in immortalized hVM1 cells.

Experimental Procedures
Cell Culture hVM1 cells were cultured on 10μg/mL polylysine (PLL)pretreated plastic ware in proliferation media (Dulbecco's modified Eagle's medium/F12 medium with epidermal growth factor and basic fibroblast growth factor (20ng/mL each; R&D System), Glutamax (Invitrogen), 1% Albumax (Invitrogen), 50mM HEPES (Invitrogen), 0.6% glucose, N2 supplement (Invitrogen), 1X nonessential amino acids, and penicillin/streptomycin). To induce cell differentiation, cells were seeded at 105 cells/cm2 in proliferation medium, and after 24 hours, the proliferation medium was replaced by differentiation medium (without epidermal growth factor and basic fibroblast growth factor and containing 1 mM dibutyryl-cAMP (Sigma) and 2ng/ml human recombinant glial cellderived neurotrophic factor (Preprotech)). Differentiation medium was changed every second day until the end of the experiment.

Cell Viability Assay
The resazurin reduction method was used for the determination of cell viability. The method is based on the reduction of resazurin, a normally non-fluorescent compound, to resofurin, a fluorescent metabolite, due to the highly reducing milieu of a living cell [34].

Determination of Target Gene Expression by Real-Time PCR
Cultures of hVM1 cells at 7 days of differentiation after exposure to 1 and 5μg/L sodium arsenite were collected and washed with PBS for RNA isolation using the Trizol method. The concentration and quality were determined by spectrophotometric analysis using a

Statistical Analysis
For

Viability Assay
The viability assay was performed in hVM1 cells at 7 days of differentiation using the resazurin method. The hVM1 cells were treated with different concentrations of sodium arsenite (5-1000μg/L) for 24 hours. We observed that only at an arsenic concentration of 5μg/L were cells able to maintain viability above 80%; this was the concentration used for subsequent experiments.
Above 10μg/L of sodium arsenic, the cell viability declined in a dose-dependent manner and was significantly lower than 80% (p<0.001) (Figure 1).

Expression of miR-132 and CREB in hVM1 cells Exposed To sodium arsenite
Once the sodium arsenite concentration at which hVM1 cells maintained greater than 80% viability was determined, we evaluated the expression of miR-132 and its transcription factor CREB in cultures exposed to 1 and 5μg/L of arsenic in both undifferentiated cultures and after 7 days of differentiation using qRT-PCR. Five independent experiments with duplicates of each condition were performed and the results are represented as the fold change between undifferentiated and differentiated cells.
Although there were no significant differences between treated and untreated cells, we observed less dispersion in the data from hVM1 cultures exposed to 5μg/L of arsenic and a trend toward decreased expression of miR-132 in cultures after 7 days of differentiation compared to untreated cells (Figure 2A). We observed that the expression of the transcription factor CREB exhibited the opposite behavior to miR-132 expression ( Figure 2B).  It has been demonstrated that dynamic changes in the expression of miRs occurs during neural differentiation, so to understand the effect of arsenic on miR-125b expression in the regulation of neural differentiation, the expression of miR-125b and a target gene (nestin) were assessed by real-time PCR. The results show that miR-125b was downregulated in both exposed cultures (1 and 5μg/L) and statistical differences were observed between untreated and arsenic exposed cultures ( Figure 3A). At the same time, nestin gene expression was enhanced, which has been described like target gene of miR-125b ( Figure 3B). We found that miR-125 and nestin gene expression showed opposing behavior, i.e. in untreated cultures, miR-125b expression was higher than in arsenic exposed cells. Moreover, and the expression of nestin was augmented in differentiation cultures following arsenic exposure, which could be a discreet effect of miR regulatory expression ( Figure 3C).

BDNF Expression in Sodium Arsenite Exposed Hvm1 Cells During Differentiation
Because BDNF is one of the most important neuromodulators of neural differentiation and function, the analysis of its expression was included in this study. We evaluated BDNF expression under differentiation conditions and in cells exposed to 1 and 5μg/L of sodium arsenite. As we expected, BDNF expression was augmented in untreated differentiation cultures, but there were no statistically significant differences when hVM1 cells were exposed to sodium arsenite during differentiation (Figure 4).  Finally, we analyzed miR-148 and DNMT3b (a target gene) expression in cells exposed to sodium arsenite during differentiation. miR-148a expression was significantly increase in cultures exposed to low doses (1μg/L) of arsenic compared to the other culture conditions, i.e. untreated and cells exposed to 5μg/L of arsenic ( Figure 5A). We expected to find greatly diminished expression of DNMT3b in the 1μg/L arsenic cultures, but DNMT3b expression in the exposed groups was not significantly different compared to the control condition. However, we observed a trend for downregulation in both exposed cultures (1 and 5μg/L) ( Figure   5B). On the other hand, transcription factor cAMP-Response Element Binding Protein (CREB) has been extensively studied in the nervous system and is associated with neuronal functions such as plasticity, neuronal maturation and membrane depolarization [35].

Discussion
CREB regulates a wide range of genes and miRs, including miR-132 [36]. miR-132 over-expression is induced by neurotrophins like BDNF through activation of the ERK1/2 pathway, implicating CREB phosphorylation by MSK1 [37]. Our results show high data dispersion and a trend toward downregulated miR-132 expression following arsenic exposure at 5μg/L; however, this was not statistically significant. Additionally, because CREB is an indirect miR-132 target, our results suggest an association between miR-132 and CREB expression. Previous reports have shown that arsenic can induce alterations in CREB on the mRNA level, although this does not necessarily suggest a similar change at the protein level [38]. In addition, the expression patterns between miR-132 and CREB observed at 5μg/L of arsenic could be a mechanism of self-regulation. [38]. proposed a model for miR-132 regulation through the suppression of p300, which is a co-activator of CREB. miR-132 expression is induced due CREB activation, then mature miR-132 is able to bind to the 3'UTR of p300 mRNA and CREB activation is suppressed, allowing for miR-132 downregulation.
Therefore, during the differentiation of hVM1 cells, sodium arsenite exposure may alter miR-132 expression and may be associated with the downregulation of CREB expression, suggesting that miR-132 is a regulator in the mechanism to balance CREB recovery on the gene expression level due to p300 regulation. Several important and novel functions of miR-125b have been reported in neural differentiation, suggesting that this is a key player in the molecular cascade that contributes to the irreversible commitment of pluripotent human stem cells to the neural lineage. Actually, at least 10 targets relevant to neural differentiation and function have been identified for the action of miR-125b [39]. One of the most important targets that have been studied is nestin [40]. which has a crucial role in neural cell differentiation. A statistically significant decrease in miR-125b expression was observed in differentiating hVM1 cells exposed to arsenic in the present study. This was correlated with nestin upregulation in a dose-dependent manner following sodium arsenite exposure. The increase in the expression of nestin in differentiating cells could be a marker of damage to the differentiation and function cellular program. Our miR-125b results reveal that the neural differentiation of hVM1 cells can be disturbed in the presence of 1 and 5μg/L arsenite, although it is important verify the neuronal phenotype, which needs to be determined in further studies. In order to elucidate other possible targets for arsenic, we analyzed BDNF expression, as it promotes neural differentiation and is implicated in miR expression [41,42] However, we did not observe a statistically significant difference between untreated and exposed cultures. Arsenic had been implicated in the disruption of cellular methylation reactions, particularly in DNA methylation Several reports have shown that arsenic exposure represses DNMT mRNA expression and activity [43]. In this study, we evaluated changes in miR-148a and DNMT3b expression and found significantly augmented miR-148 expression in hVM1 cultures exposed to 1μg/L of arsenic, the effect was the opposite at 5μg/L of arsenic as hormesis effect.
Furthermore, DNMT3b expression was not significantly different compared to the unexposed group, although a trend toward downregulation in hVM1 cells exposed to both concentrations of arsenic appeared, as previously described by [13] They observed that human keratinocyte-derived HaCat cells exposed to arsenic up to 5μM for 72 h had significantly inhibited expression of DNMT1 and DNMT3a. Diminished DNMT expression could be indicated in global and gene-specific DNA hypomethylation, although the CpG methylation status not assayed. These results suggest that miR-148 is not the only regulator of DNMT3b expression, accordingly, as has been reported previously The effects of arsenic on miR expression described in this study could be an important linkage between wide physiologic alterations and a common mechanism. miRNA is considered to be a master regulator of several cellular process, including neural differentiation, hence it is possible to suggest as a likely mechanism arsenic-mediated changes to gene expression through the disruption of several signaling pathways.
In agreement with our study, [44] showed that sodium arsenite affects the signaling pathways that control neural differentiation of human neural stem cells through overactivation of MEK-ERK and suppression of PI3K-AKT. Because multiple key transcription factors are affected, changes in miR and target gene expression could occur.

Conclusion
In conclusion, these findings show that sodium arsenite exposure promotes changes in miR expression, which affect important neural differentiation regulators and therefore could affect the differentiation of hVM1 cells.