MicroRNA and messenger RNA profiling reveals new biomarkers and mechanisms for RDX induced neurotoxicity
© Deng et al.; licensee BioMed Central Ltd. 2014
Published: 16 December 2014
RDX is a well-known pollutant to induce neurotoxicity. MicroRNAs (miRNA) and messenger RNA (mRNA) profiles are useful tools for toxicogenomics studies. It is worthy to integrate MiRNA and mRNA expression data to understand RDX-induced neurotoxicity.
Rats were treated with or without RDX for 48 h. Both miRNA and mRNA profiles were conducted using brain tissues. Nine miRNAs were significantly regulated by RDX. Of these, 6 and 3 miRNAs were up- and down-regulated respectively. The putative target genes of RDX-regulated miRNAs were highly nervous system function genes and pathways enriched. Fifteen differentially genes altered by RDX from mRNA profiles were the putative targets of regulated miRNAs. The induction of miR-71, miR-27ab, miR-98, and miR-135a expression by RDX, could reduce the expression of the genes POLE4, C5ORF13, SULF1 and ROCK2, and eventually induce neurotoxicity. Over-expression of miR-27ab, or reduction of the expression of unknown miRNAs by RDX, could up-regulate HMGCR expression and contribute to neurotoxicity. RDX regulated immune and inflammation response miRNAs and genes could contribute to RDX- induced neurotoxicity and other toxicities as well as animal defending reaction response to RDX exposure.
Our results demonstrate that integrating miRNA and mRNA profiles is valuable to indentify novel biomarkers and molecular mechanisms for RDX-induced neurological disorder and neurotoxicity.
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), is a well known contaminant to territory, soil and ground water due to military and manufacturing activities. A series of studies have demonstrated that RDX can cause neurotoxicity including seizure in human and animals [1–4]. RDX can also induce immunotoxicity in rats [5–7].
While many effects of RDX exposure are known, the molecular mechanisms of RDX caused toxicity have not been well characterized. It appears that RDX binding to the GABAA receptor convulsant site maybe the primary mechanism of seizure induction by RDX and that reduction of GABAergic inhibitory transmission in the amygdala is involved in the generation of RDX-induced seizures[8–10]. But more mechanisms still needed to be studied such as epigenetic mechanisms.
Recently, It was  found RDX exposure could significantly alter a large number of miRNA expression in mouse brain and liver tissues with a 28 day long term exposure. MiRNAs are endogenous, small non-coding RNAs, usually 18-25 nucleotides long, have been found to play crucial roles in post-transcriptionally regulation of mRNA. MiRNAs have been found to involve in almost all fundamental important biological processes and diseases including neurological diseases and nervous system function . MiRNAs carries out its function by specifically binding 3'UTR of mRNA to interrupt mRNA translation or cause degradation of transcripts [13–15]. Recent reports suggest that miRNA could play an opposite role by activating a gene expression at certain conditions [16, 17].
We use rat as a model species to investigate the impact of RDX on both miRNA and mRNA expression in rat brain tissues with a sub-acute short term exposure (48 h). The objectives of the study are to see whether we could see an across species conserved miRNA expression between rat and mouse, find overlapped targets between the putative targets of regulated miRNAs and mRNA genes regulated by RDX, identify early expression altered miRNAs and genes as new markers for assessing RDX induced neurotoxicity, and further understand the molecular mechanisms of RDX caused neurotoxicity. Since that miRNAs are highly conserved between humans and rats, this study should improve our understanding of the molecular mechanisms of RDX induced neurological disorders and diseases.
There are still very few studies to use miRNA expression profiles for characterizing RDX caused toxicity. There is no report to integrate RDX altered miRNA and mRNA expression profiles.
Materials and methods
RDX (purity > 99%) was obtained from Stan Caulder (Naval Surface Warfare Center, Indianhead, MD, USA).
Animals and Treatment
Female Sprague-Dawley rats (175-225 grams) were from the in-house breeding colony (College of Pharmacy, University of Louisiana at Monroe [ULM] and treated in accordance with the Guide for Use and Care of Animals . Breeders were from Harlan-Sprague Dawley in Madison, WI. Housing consisted of a 12 h light/dark cycle with ad libitum access to tap water and rodent chow (Harlan/Teklad 7012, Madison, WI). Rats were housed individually in polycarbonate cages on hardwood bedding (Sani-chips, Harlan/Tekland, Madison, WI) one week prior to treatment. Food was withdrawn the night before treatments, which were administered by gavage between 8 and 10 AM. Study protocols were preapproved by the Institutional Animal Care and Use Committee of the University of Louisiana at Monroe (Animal Welfare Assurance Number A3641-01).
Groups of rats were weighed and randomly assigned to treatment. Treatments were vehicle (5% v/v DMSO in corn oil), RDX (47 mg/kg). Rats were observed continuously for the first hour after dosing, hourly for 8 h and daily thereafter. Moribund rats were euthanized with CO2. At 48 hours after treatment, survivors were anesthetized with CO2. A portion of the brain was removed and flash frozen in liquid N2 and stored at -70º C for miRNA and mRNA microarray analyses.
Total RNA extraction
Total RNA was extracted from about 30 mg of brain tissue. Tissues were homogenized in the lysis buffer with FAST Prep-24 from MP before using RNeasy kits (Qiagen). Total RNA concentrations were measured using NanoDrop® ND-1000 Spectrophotometer (NanoDrop technologies, Wilmington, DE, USA). The integrity and quality of total RNA was checked on an Agilent 2100 Bioanalyzer (Palo Alto, CA). The gel-like images generated by the Bioanalyzer show that total RNAs have two bands, represent 18S and 26S RNA of mammalian RNA. Nuclease-free water (Ambion) was used to elute total RNA.
We extracted total miRNA from each sample using the mirVana miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's instructions. Briefly, 0.03-0.05 g tissues were weighed and placed in a new 2-mL micro-centrifuge tube, followed by adding 300 μL lysis/binding buffer. Then, the tissues were thoroughly disrupted and homogenized using a Sonic Dismembrator (model 100, Fisher Scientific, Atlanta, GA). After homogenization, we added 30 μL miRNA homogenate additive to each tissue lysate, vortexed it for 10 sec, and then incubated it on ice for 10 min. After washing, we eluted the total RNAs using 100 μL elution buffer provided in the miRNA isolation kit. We performed all these operations on ice. The extracted RNA was quantified using a NanoDrop ND-100 spectrophotometer (NanoDrop Technologies, Wilmington, DE), aliquoted, and immediately stored at −80°C until analysis.
MicroRNA microarray hybridization
The miRNA micro-array analysis was performed by LC Sciences (Houston, TX) as described previously . Briefly, the assay started with approximately 6 μg total RNA. After the total RNAs were fractionated by size using a YM-100 Microcon centrifugal filter (Millipore, Billerica, MA), poly(A) tails were added to RNA sequences with lengths less than 300 nucleotides using poly(A) polymerase. Then, an oligonucleotide tag was ligated to the poly(A) tail for later fluorescent dye staining. RNA samples from brain extracts were hybridized overnight using two different tags on a μParaflo microfluidic chip using a microcirculation pump developed by Atactic Technologies (Houston, TX).
mRNA microarray hybridization
mRNA microarray hybridization was done as described previously. Briefly cDNA from 1 ug total RNA was synthesized, hybridized to arrays, and detected by secondary hybridization to Alexa647 and Cy3 dendrimer oligonucleotides using an Array900 detection kit per manufacturer's instructions (Genisphere, Hatfield, PA). cDNA was hybridized to 8 K Sigma/Compugen rat 70-mer oligonucleotide libraries arrayed on glass slides (Center for Applied Genomics, Newark, NJ http://www.cag.icph.org/).
MiRNA microarray data analysis
MiRNA microarray was normalized by Lowess normalization algorithm using GeneSpring 10.0 (Agilent Technologies, Foster City, CA, USA). We first filtered substance according to flags. All substances that were at least 50% of samples in any of 2 out of 2 conditions (control group and RDX treatment group) have present calls according to the feature extraction software data will be remained. 215 substances were left after the flag filtering. We also judged a miRNA detectable using at least three criteria: a) signal intensity bigger than three times background standard deviation; b) spot coefficient of variance (CV) < 0.5, in which we calculated CV as (standard deviation)/(signal intensity); and c) at least two spots of four technical repeats in each chip and two of three biological replicates in either control or treatment group have signal greater than three times background standard deviation. We found that 178 miRNAs could be detected in either control or treatment group, and were also in the 215 miRNA list. Therefore, 178 detected miRNAs were employed to identify differentiated miRNAs in rat brain after exposure to RDX. Differentiated miRNAs were computed using un-paired T-test with a cut off p value <= 0.05 and fold change >=1.2 (corrected by Benjamini Hochberg at a FDR <= 0.05).
MRNA microarray data analysis
MRNA microarray data analysis was done as we described previously [20, 21]. Briefly, the data was normalized based on Lowess normalization method, Bayesian statistical analysis with 5% FDR was used to identify differential genes between two groups.
MiRNA target prediction
Reverse-transcription quantitative PCR (QRT-PCR)
We selected miRNAs with aberrant expression in microarray analysis and then validated them using qRT-PCR on an ABI7300 system (Applied Biosystems, Foster City, CA). We used TaqMan miRNA assays to detect and quantify mouse miRNAs using stem-loop RT-PCR according to the manufacturer's instructions.
Gene functional analysis and network construction
Physiological process and pathway analyses were performed using the Ingenuity pathway analysis (IPA) tool. A physiological process or a pathway with an enrichment p value <= 0.05 was considered to be significant (Ingenuity Systems, Inc., Redwood City, CA). Gene networks were constructed based on the IPA tool. A score was assigned to a network according to the fit of the original set of significant genes. This score reflects the negative logarithm of the p value that indicates the likelihood of the focus genes in a network being found together due to random chance .
Regulation of miRNA expression in the brain tissues of rats exposed to RDX
Significantly regulated miRNAs in rat brain after exposure to RDX.
Computational predictions of the putative targets of regulated miRNAs
Figure 3B exhibited top 10 significant canonical pathways enriched in the 1589 target genes. Among them, two canonical pathways axonal guidance signaling and reelin signaling in neurons were clearly involved in nervous system function. Seventy six target genes (Additional file 2) fell into the axonal signaling pathway which is related to neurotransmitters and other nervous System Signaling. Twenty-two genes (Additional file 2) play a role in reelin signaling in neurons pathway.
Comparison of miRNA target genes and mRNA expression profile
Overlapped genes between putative target genes of miRNAs and differential mRNA genes regulated by RDX.
Entrez Gene Name
lipopolysaccharide-induced TNF factor
neuronal pentraxin II
solute carrier family 38, member 2
VGF nerve growth factor inducible
BTG3 associated nuclear protein
chromosome 5 open reading frame 13
centrosomal protein 350 kDa
family with sequence similarity 82, member A2
polymerase (DNA-directed), epsilon 4 (p12 subunit)
Rho-associated, coiled-coil containing protein kinase 2
solute carrier family 35, member E4
The relationship between miRNAs and their target genes regulated by RDX.
KIAA1033, CEP350, ROCK2
BANP, CALD1, POLE4
HMGCR, LITAF, NPTX2
CALD1, C5orf13, KIAA1033
POLE4, SLC35E4, C5orf13
Over half (8) of the overlapped target genes are involved in neurological disease and nervous system function.
granular cell tumor
BANP, C5ORF13, HMGCR, ROCK2
BANP, C5ORF13, HMGCR, LITAF, POLE4, ROCK2
migration of brain cancer cell lines
Nervous System Development and Function
morphology of dendritic spines
Nervous System Development and Function
sprouting of neurites
Nervous System Development and Function
differentiation of neurons
Nervous System Development and Function
retraction of neurites
Comparative pathway and network analysis
Verification of miRNA microarray responses using real time QRT-PCR
MiRNA alteration in response to RDX exposure
This is the first report that comprehensively analyzes the rat brain miRNA expression profile in response to RDX exposure. We found that 9 miRNAs whose expression was altered in rat brain tissues by RDX. Among them, 6 miRNAs were up-regulated and 3 miRNAs were down-regulated. Two miRNAs miR-98 and miR-7a were induced in rat brain tissues after exposure to RDX, and they were also up-regulated in mouse brain tissues treated with RDX . MiR-27b and miR-320 were up-regulated and down-regulated respectively in our study, but not significantly changed in mouse brain tissue. However they were altered in mouse liver tissues with the same direction as rat brain tissues by RDX. Some of our regulated miRNA are not shown in Zhang's regulated miRNA lists, and also RDX induced much more miRNAs in mouse brain than our case. This should not be hard to explain, because Zhang's study is a long term exposure (28 days), we only used 2 day short exposure. It may also have dose and species specific responses.
Some of these 9 regulated miRNAs have been shown to participate in neurological diseases and neural system function. For instance, miR-7a was reported to be involved in glioblastoma  and Parkinson's disease . MiR-27a and MiR-129 were shown to play a role in autism spectrum disorder (ASD) . MiR-320 was observed to be involved in neurodegenration  and retinoblastoma . MiR-342-3p has been found to play a role in neurodegenration  and prion disease . Our results indicate that RDX could induce neurological diseases and neurotoxicity through regulating these miRNAs.
The putative targets of RDX regulated miRNAs involved in neurotoxicity
Existing studies have exhibited that RDX exposure induced adverse central nervous system (CNS) syndromes such as convulsion, epileptic seizure, and loss of reflexes in human and experimental animals [1, 5–7]. However, how RDX causes neurotoxicity at molecular level is not well known. Through the functional analyses of these target genes, we found that multiple evidences to support that RDX affects neural system development and neurological pathways. Nervous system development and function is in the top significant physiological system development and function category (Figure 3A). Axonal guidance signaling pathway is in the top 2 significantly pathways enriched in the canonical pathway analysis (Figure 3B). The pathway plays a crucial role in neuronal connections that are formed by the extension of axons, which migrate to reach their synaptic targets. The axonal growth cone, located at the axon leading edge, contains receptors that sense attractive and repulsive guidance cues, which help navigate the axon to its final destination [31–35].
Another important pathway in the top significant pathway list (Figure 3B) is reelin signaling in neurons. Reelin is a large extracellular glycoprotein involved in the development of architectonic patterns, and Cajal-Retzius cells of the human embryonic marginal zone are the primary site of synthesis for reelin. In the hippocampus, reelin also regulates the growth and/or distribution of afferent entorhinal and commissural axons. The genes in the pathway provide molecular mechanisms that control brain development and, potentially, the pathogenesis of neurodegenerative disorders [36–38]. Moreover, the most significant gene network based on the putative target genes is involved in neural system function and development (Figure 4). Regulating the gene network and the pathways could partially explain the molecular mechanism for RDX induced neurotoxicity.
Overlapped genes between putative targets of regulated miRNAs and differential mRNA genes revealing new markers for RDX induced neurotoxicity
We found 15 overlapped genes, which are about 12% of differential mRNA genes (123 genes). The number is not big and the percentage is similar to other studies. For instance, the overlapped target genes was 12% in prion induced miRNA and mRNA profiles . The Several reasons could account for the small portion of overlapping. First, the rat mRNA array is not the whole genome array, only about one third of the genome (8000 probes) is used, but the prediction of miRNA targets is based on the whole genome. Second, the prediction is static, which means the targets are predicted based on any condition, but the mRNA genes are differentially expressed at a certain condition. Third, the predictive putative targets may contain many false positives. Fourth, technical limitation for both miRNA and mRNA arrays may also leave out potential overlapped genes.
MiRNAs mediated gene network of immune and inflammation response genes regulated by RDX
Immune and inflammation response genes are highly enriched in differentially expressed mRNAs after exposure to RDX, but not in the putative target genes of regulated miRNAs. Interestingly, the immune and inflammation response genes connected network (Figure 6) indicates that RDX could first modulate miRNA expression and then trigger an immune and inflammation gene network. For instance, miR-98 could target IL10, which then up-regulate TNF, which induces the expression of the transcription factor HSF1 (Figure 6), to carry out immune functions. RDX regulated immune response could contribute to RDX induced neurotoxicity and other toxicities as well as animal defending reaction response to RDX exposure.
Our results demonstrate that integrating miRNA and mRNA profiles is valuable to indentify novel biomarkers and molecular mechanisms for RDX-induced neurological disorder and neurotoxicity. RDX could first induce the expression of miR-71, miR-27ab, miR-98, and miR-135a, then reduce the expression of POLE4, C5ORF13, SULF1 and ROCK2, and finally induce neurotoxicity, which provides an alternative mechanism to explain RDX induced neurotoxicity.
This work and its publication were supported by research grants from Natural Science Foundation Hubei Province of China (2011CDB236) and United States National Institutes of Health (NIH). MQY was supported by NIH/NIGMS 5P20GM10342913 and ASTA award # 15-B-23. YD was supported by NIH/NCI R21CA164764.
This article has been published as part of BMC Genomics Volume 15 Supplement 11, 2014: Selected articles from the 2014 International Conference on Advances in Big Data Analytics. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcgenomics/supplements/15/S11.
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