Revisiting Chaos Theorem to Understand the Nature of miRNAs in Response to Drugs of Abuse.

Just like Matryoshka dolls, biological systems follow a hierarchical order that is based on dynamic bidirectional communication among its components. In addition to the convoluted inter-relationships, the complexity of each component spans several folds. Therefore, it becomes rather challenging to investigate phenotypes resulting from these networks as it requires the integration of reductionistic and holistic approaches. One dynamic system is the transcriptome which comprises a variety of RNA species. Some, like microRNAs, have recently received a lot of attention. miRNAs are very pleiotropic and have been considered as therapeutic and diagnostic candidates in the biomedical fields. In this review, we survey miRNA profiles in response to drugs of abuse (DA) using 118 studies. After providing a summary of miRNAs related to substance use disorders (SUD), general patterns of miRNA signatures are compared among studies for single or multiple drugs of abuse. Then, current challenges and drawbacks in the field are discussed. Finally, we provide support for considering miRNAs as a chaotic system in normal versus disrupted states particularly in SUD and propose an integrative approach for studying and analyzing miRNA data.

Chronic nicotine exposure systemically alters microRNA expression profiles during post-embryonic stages in Caenorhabditis elegans.

Tobacco smoking is associated with many diseases. Addiction is of the most notorious tobacco-related syndrome and is mainly attributed to nicotine. In this study, we employed Caenorhabditis elegans as a biological model to systemically investigate the effect of chronic nicotine exposure on microRNA (miRNA) expression profile and their regulated biochemical pathways. Nicotine treatment (20 µM and 20 mM) was limited to the post-embryonic stage from L1 to L4 (∼31 h) period after which worms were collected for genome-wide miRNA profiling. Our results show that nicotine significantly altered the expression patterns of 40 miRNAs. The effect was proportional to the nicotine dose and was expected to have an additive, more robust response. Based on pathway enrichment analyses coupled with nicotine-induced miRNA patterns, we inferred that miRNAs as a system mediates "regulatory hormesis", manifested in biphasic behavioral and physiological phenotypes. We proposed a model where nicotine addiction is mediated by miRNAs' regulation of fos-1 and is maintained by epigenetic factors. Thus, our study offers new insights for a better understanding of the sensitivity of early developmental stages to nicotine.

High-throughput deep sequencing shows that microRNAs play important roles in switchgrass responses to drought and salinity stress.

MicroRNAs (miRNAs) are an important class of small regulatory RNAs. The goal of this study was to analyse stress-responsive miRNAs in switchgrass (Panicum virgatum), the emerging biofuel crop, to facilitate choosing gene targets for improving biomass and biofuel yield. After sequencing three small RNA libraries constructed from control, salt- and drought-treated switchgrass using Illumina sequencing technology, we identified 670 known miRNA families from a total of more than 50 million short reads. A total of 273 miRNAs were identified with precursors: 126 conserved miRNAs and 147 novel miRNAs. Of them, 265 miRNAs were found to have their opposite sequences (miRNA*) with 2-nt overhang on the 3' end. Of them, 194 were detected in switchgrass transcriptome sequences generated from 31 high-throughput RNA sequencing (RNA-Seq) data sets in NCBI. Many miRNAs were differentially or uniquely expressed during salinity or drought stress treatment. We also discovered 11 miRNA clusters containing 29 miRNAs. These identified miRNAs potentially targeted 28549 genes with a various function, including transcription factors, stress-response proteins and cellulose biosynthesis-related proteins. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the identified miRNAs and their targets were classified to 3779 GO terms including 1534 molecular functions, 1851 biological processes and 394 cellular components and were enriched to 147 KEGG pathways. Interestingly, 195 miRNA families and 450 targets were involved in the biosynthesis pathways of carbon, glucose, starch, fatty acid and lignin and in xylem formation, which could aid in designing next-generation switchgrass for biomass and biofuel.

Evaluation and identification of reliable reference genes for toxicological study in Caenorhabditis elegans.

To identify reliable reference genes for toxicological studies, 16 commonly-used reference genes were selected as candidates to evaluate their expression stabilities under experimental conditions in Caenorhabditis elegans. Sixteen candidates were composed of 12 protein-coding genes and 4 non-coding RNAs, they were act-2, ama-1, arp-6, cdc-42, csq-1, eif-3.C, idhg-1, mdh, pmp-3, rbd-1, tba-1, Y45F10D.4, 18S rRNA, Ce234, U18, and U6. Larval stage 1 synchronized hermaphrodites were exposed to benzo-α-pyrene (BαP), chlorpyrifos, diazinon, gossypol, zinc oxide nanoparticles, and the vehicle control DMSO for 30 h, respectively. Expression stabilities of candidate genes were analyzed using 4 independent evaluating approaches (BestKeeper, the delta Ct approach, geNorm, and NormFinder) followed by a comprehensive method. Results showed that there were slight differences in ranking order between evaluation methods due to their different assumptions and computations. The results also showed that responses of candidate genes to different chemicals were distinct, 18S rRNA was the best for BαP and chlorpyrifos, tba-1 was the most stable gene for diazinon and gossypol treatments, while pmp-3 was more stable for zinc oxide exposure. Additionally, results demonstrated that combinations of multiple genes were more reliable than individual gene, suggesting selecting two or more candidates as reference genes may generate more reliable results for toxicological studies.

Dose-Dependent Effects of GLD-2 and GLD-1 on Germline Differentiation and Dedifferentiation in the Absence of PUF-8.

PUMILIO/FBF (PUF) proteins have a conserved function in stem cell regulation. Caenorhabditis elegans PUF-8 protein inhibits the translation of target mRNAs by interacting with PUF binding element (PBE) in the 3' untranslated region (3' UTR). In this work, an in silico analysis has identified gld-2 [a poly(A) polymerase] as a putative PUF-8 target. Biochemical and reporter analyses showed that PUF-8 specifically binds to a PBE in gld-2 3' UTR and represses a GFP reporter gene carrying gld-2 3' UTR in the C. elegans mitotic germ cells. GLD-2 enhances meiotic entry at least in part by activating GLD-1 (a KH motif-containing RNA-binding protein). Our genetic analyses also demonstrated that heterozygous gld-2(+/-) gld-1(+/-) genes in the absence of PUF-8 are competent for meiotic entry (early differentiation), but haplo-insufficient for the meiotic division (terminal differentiation) of spermatocytes. Indeed, the arrested spermatocytes return to mitotic cells via dedifferentiation, which results in germline tumors. Since these regulators are broadly conserved, we thus suggest that similar molecular mechanisms may control differentiation, dedifferentiation, and tumorigenesis in other organisms, including humans.

Triclosan Disrupts SKN-1/Nrf2-Mediated Oxidative Stress Response in C. elegans and Human Mesenchymal Stem Cells.

Triclosan (TCS), an antimicrobial chemical with potential endocrine-disrupting properties, may pose a risk to early embryonic development and cellular homeostasis during adulthood. Here, we show that TCS induces toxicity in both the nematode C. elegans and human mesenchymal stem cells (hMSCs) by disrupting the SKN-1/Nrf2-mediated oxidative stress response. Specifically, TCS exposure affected C. elegans survival and hMSC proliferation in a dose-dependent manner. Cellular analysis showed that TCS inhibited the nuclear localization of SKN-1/Nrf2 and the expression of its target genes, which were associated with oxidative stress response. Notably, TCS-induced toxicity was significantly reduced by either antioxidant treatment or constitutive SKN-1/Nrf2 activation. As Nrf2 is strongly associated with aging and chemoresistance, these findings will provide a novel approach to the identification of therapeutic targets and disease treatment.

Benzo-α-pyrene induced oxidative stress in Caenorhabditis elegans and the potential involvements of microRNA.

In the present study oxidative stress induced by Benzo-α-pyrene (BaP) exposure and the potential involvements of microRNA were investigated. The Caenorhabditis elegans (C. elegans) was applied as model organism. The C. elegans at L1-stage were randomly divided into 4 groups and exposed to 0, 0.2, 2.0, and 20µM BaP for 30h. Expressions of SKiNhead-1 (SKN-1), gamma-glutamine cysteine synthase heavy chain (GCS-1), and their potential regulatory factors in insulin/IGF-1/FOXO signaling pathway and the p38 MAPK pathway were analyzed. The expressions of potentially involved microRNAs were investigated as well. Results demonstrated that expressions of SKN-1 and GCS-1 were altered significantly following BaP exposure (P<0.05). Meanwhile, expressions of multiple related factors were changed after BaP treatments. The altered factors include AKT-1, DAF-16, glutathione synthetase (GSS-1), glutathione S-transferase-24 (GST-24), mitogen-activated protein kinase kinase-4 (MKK-4), multidrug resistance-associated protein-1 (MRP-1), and pyruvate dehydrogenase kinase-2 (PDHK-2) (P<0.05). In addition, results showed that exposure to BaP led to altered expressions of microRNA. Out of the 28 tested microRNAs, expressions of miR-1, miR-355, miR-50, miR-51, miR-58, miR-796, miR-797, and miR-84 were modified. Findings of the present study include that BaP exposure caused oxidative stress in C. elegans. The expressional response of GCS-1 to BaP exposure might be independent of the regulation of SKN-1 in C. elegans. The microRNAs might be involved in the regulations of SKN-1 and GCS-1 expression following BaP exposure in C. elegans.

A comprehensive approach to identify reliable reference gene candidates to investigate the link between alcoholism and endocrinology in Sprague-Dawley rats.

Gender and hormonal differences are often correlated with alcohol dependence and related complications like addiction and breast cancer. Estrogen (E2) is an important sex hormone because it serves as a key protein involved in organism level signaling pathways. Alcoholism has been reported to affect estrogen receptor signaling; however, identifying the players involved in such multi-faceted syndrome is complex and requires an interdisciplinary approach. In many situations, preliminary investigations included a straight forward, yet informative biotechniques such as gene expression analyses using quantitative real time PCR (qRT-PCR). The validity of qRT-PCR-based conclusions is affected by the choice of reliable internal controls. With this in mind, we compiled a list of 15 commonly used housekeeping genes (HKGs) as potential reference gene candidates in rat biological models. A comprehensive comparison among 5 statistical approaches (geNorm, dCt method, NormFinder, BestKeeper, and RefFinder) was performed to identify the minimal number as well the most stable reference genes required for reliable normalization in experimental rat groups that comprised sham operated (SO), ovariectomized rats in the absence (OVX) or presence of E2 (OVXE2). These rat groups were subdivided into subgroups that received alcohol in liquid diet or isocalroic control liquid diet for 12 weeks. Our results showed that U87, 5S rRNA, GAPDH, and U5a were the most reliable gene candidates for reference genes in heart and brain tissue. However, different gene stability ranking was specific for each tissue input combination. The present preliminary findings highlight the variability in reference gene rankings across different experimental conditions and analytic methods and constitute a fundamental step for gene expression assays.

Nicotine exposure and transgenerational impact: a prospective study on small regulatory microRNAs.

Early developmental stages are highly sensitive to stress and it has been reported that pre-conditioning with tobacco smoking during adolescence predisposes those youngsters to become smokers as adults. However, the molecular mechanisms of nicotine-induced transgenerational consequences are unknown. In this study, we genome-widely investigated the impact of nicotine exposure on small regulatory microRNAs (miRNAs) and its implication on health disorders at a transgenerational aspect. Our results demonstrate that nicotine exposure, even at the low dose, affected the global expression profiles of miRNAs not only in the treated worms (F0 parent generation) but also in two subsequent generations (F1 and F2, children and grandchildren). Some miRNAs were commonly affected by nicotine across two or more generations while others were specific to one. The general miRNA patterns followed a "two-hit" model as a function of nicotine exposure and abstinence. Target prediction and pathway enrichment analyses showed daf-4, daf-1, fos-1, cmk-1, and unc-30 to be potential effectors of nicotine addiction. These genes are involved in physiological states and phenotypes that paralleled previously published nicotine induced behavior. Our study offered new insights and further awareness on the transgenerational effects of nicotine exposed during the vulnerable post-embryonic stages, and identified new biomarkers for nicotine addiction.

Determination of reliable reference genes for multi-generational gene expression analysis on C. elegans exposed to abused drug nicotine.

RATIONAL: More research has recently been focused on multigenerational toxicogenomics impacts. Such studies rely on behavioral as well as genetic and epigenetic analyses using various biotechniques. Of these technologies, quantitative reverse transcriptase PCR is considered as a mature discovery and validation tool. Nevertheless, the interpretation of the resulting gene expression necessitates the establishment of reliable internal controls for normalization. No study has been performed to identify reliable reference genes in multigenerational settings. OBJECTIVES: The primary aim was to evaluate the stability of 16 reference gene candidates in Caenorhabditis elegans exposed to nicotine and their two subsequent generations for determining the most reliable reference genes for multigenerational study. METHODS: We exposed C. elegans to nicotine in the F0 generation and investigated the relative stabilities of 16 housekeeping genes in L4 larvae across three generations (F0, F1, and F2) using five statistical approaches (geNorm, ∆Ct method, NormFinder, BestKeeper, and RefFinder). RESULTS: geNorm shows that CDC-42 and Y45F10D.4 were the most stable reference genes. Based on NormFinder, TBA-1, EIF3.C, ARP-6, CDC-42, and MDH2 may serve as the top reliable reference genes. Comparative ∆Ct method ranked TBA-1, CDC-42, EIF3.C, ARP-6, and Y45F10D.4 as the most stable reference genes. BestKeeper shows that Y45F10D.4, F35G12.2, TBA-1, CDC-42, and CSQ-1were better reference genes. Overall, TBA-1, CDC-42, EIF3.C, ARP-6, and Y45F10D.4 were the most reliable reference genes for mutigenerational nicotine-exposed study. CONCLUSIONS: Of the 16 tested gene candidates, TBA-1 and CDC-42 were the two most stable reference genes for performing reliable gene expression normalization in the multigenerational impact of nicotine exposure.

Nicotine exposure caused significant transgenerational heritable behavioral changes in Caenorhabditis elegans.

Passive and active exposure to tobacco smoking among youth is directly associated with immediate as well as long-term health deterioration. Despite all public health policies and efforts, the percentage of teenage smokers is still relatively high, especially in developing countries. Very few, if any, studies have been done on the transgenerational effect of nicotine exposed during the more sensitive, early developmental stages. We employed C. elegans as a biological model to study the multigenerational impact of chronic nicotine exposure. Nicotine treatment was limited to N2 hermaphrodites of the F0 generation. Exposure was limited to the larval period L1-L4 (~31 hours) after which worms were transferred to a fresh NGM plate. N2 hermaphrodites at L4 developmental stage were used for behavioral analysis across three generations: F0, F1, and F2. Our results show that nicotine was associated with changes in sinusoidal locomotion, speed, and body bends in L4 larvae in all three tested generations. These behavioral alterations were not restricted to F0, but were observed in F1 and F2 generations which were never exposed to nicotine. Our study is the first to reveal that nicotine addiction is heritable using C. elegans as a model organism. These results underscored the sensitivity of early development stages, with hope to spread more awareness to encourage the avoidance of nicotine exposure, especially at a young age.