MicroRNA expression changes during zebrafish development induced by perfluorooctane sulfonate

Perfluorooctane sulfonate (PFOS), a kind of widely distributed environmentally organic compound, has been found to cause developmental toxicity. Although microRNAs (miRNAs) play an important role in many metabolic tasks, whether and how they are involved in the process of PFOS‐induced toxicity is largely unknown. To address this problem, PFOS‐induced changes in miRNAs and target gene expression in zebrafish embryos, and the potential mechanism of PFOS‐induced toxic action were studied in this research. Zebrafish embryos were exposed to 1 µg ml^?1 PFOS or DMSO control from 6 h post‐fertilization (hpf) to 24 or 120 hpf. Subsequently, RNA was isolated from the embryo pool and the expression profiles of 219 known zebrafish miRNAs were analyzed using microarray. Finally, quantitative real‐time polymerase chain reaction was used to validate several miRNAs expression of microarray data. The analysis revealed that PFOS exposure induced significant changes in miRNA expression profiles. A total of 39 and 81 miRNAs showed significantly altered expression patterns after PFOS exposure 24 and 120 hpf. Of the changed miRNAs, 20 were significantly up‐regulated and 19 were significantly down‐regulated (p < 0.01) at 24 hpf, whereas 41 were significantly up‐regulated and 40 were significantly down‐regulated (p < 0.01) at 120 hpf. These miRNAs were involved in development, apoptosis and cell signal pathway, cell cycle progression and proliferation, oncogenesis, adipose metabolism and hormone secretion, whereas there is still little functional information available for 32 miRNAs. Our results demonstrate that PFOS exposure alters the expression of a suite of miRNAs and may induce developmental toxicity. Copyright © 2010 John Wiley & Sons, Ltd.     

Integrated mRNA and micro RNA profiling reveals epigenetic mechanism of differential sensitivity of Jurkat T cells to AgNPs and Ag ions

In our previous in vitro study of the toxicity on silver nanoparticles (AgNPs), we observed a dramatically higher sensitivity of Jurkat T cells to AgNPs than to Ag ions, and DNA damage and apoptosis were found to be involved in that toxicity. In this study, to understand underlying mechanism of different sensitivity of Jurket T cells to AgNPs and Ag ions, mRNA microarray and micro RNA microarray were concomitantly conducted on AgNPs and Ag ions exposed Jurkat T cells. Surprisingly only a small number of genes were differentially expressed by exposure to each of the silver (15 altered mRNA by AgNPs exposure, whereas 4 altered mRNA by Ag ions exposure, as determined 1.5-fold change as the cut-off value). miRNA microarray revealed that the expression of 63 miRNAs was altered by AgNPs exposure, whereas that of 32 miRNAs was altered by Ag ions exposure. An integrated analysis of mRNA and miRNA expression revealed that the expression of hsa-miR-219-5p, was negatively correlated with the expression of metallothionein 1F (MT1F) and tribbles homolog 3 (TRIB3), in cells exposed to AgNPs; whereas, the expression of hsa-miR-654-3p was negatively correlated with the expression of mRNA, endonuclease G-like 1 (EDGL1) in cells exposed to Ag ions. Network analysis were further conducted on mRNA-miRNA pairs, which revealed that miR-219-5p–MT1F and –TRIB3 pairs by AgNPs are being involved in various cellular processes, such as, oxidative stress, cell cycle and apoptosis, whereas, miR-654-3p and ENDOGL1 pair by Ag ions generated a much simpler network. The putative target genes of AgNPs-induced miR-504, miR-33 and miR-302 identified by Tarbase 6.0 are also found to be involved in DNA damage and apoptosis. These results collectively suggest that distinct epigenetic regulation may be an underlying mechanism of different sensitivity of Jurkat T cells to AgNPs and Ag ion. Further identification of putative target genes of DE miRNA by AgNPs and Ag ions may provide additional clues for the mechanism of differential toxicity. Overall results suggest that epigenetic mechanism is involved in toxicity of AgNPs and Ag ions in Jurkat T cells.     

Monocyte exposure to fine particulate matter results in miRNA release: A link between air pollution and potential clinical complication

Chronic exposure to PM_2.5 contributes to the pathogenesis of numerous disorders, although the underlying mechanisms remain unknown. The study investigated whether exposure of human monocytes to PM_2.5 is associated with alterations in miRNAs. Monocytes were exposed in vitro to PM_2.5 collected during winter and summer, followed by miRNA isolation from monocytes. Additionally, in 140 persons chronically exposed to air pollution, some miRNA patterns were isolated from serum seasonally. Between-season differences in chemical PM_2.5 composition were observed. Some miRNAs were expressed both in monocytes and in human serum.MiR-34c-5p and miR-223-5p expression was more pronounced in winter. Bioinformatics analyses showed that selected miRNAs were involved in the regulation of several pathways. The expression of the same miRNA species in monocytes and serum suggests that these cells are involved in the production of miRNAs implicated in the development of disorders mediated by inflammation, oxidative stress, proliferation, and apoptosis after exposure to PM_2.5.     

Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen

Aniline exposure is associated with toxicity to the spleen, however, early molecular events in aniline-induced cell cycle progression in the spleen remain unknown. MicroRNAs (miRNAs) have been implicated in tumor development by modulating key cell cycle regulators and controlling cell proliferation. This study was, therefore, undertaken on the expression of miRNAs, regulation of cyclins and cyclin-dependent kinases (CDKs) in an experimental condition that precedes a tumorigenic response. Male SD rats were treated with aniline (1?mmol/kg/day by gavage) for 7?days, and expression of miRNAs, cyclins and CDKs in rat spleens were analyzed. Microarray and/or qPCR analyses showed that aniline exposure led to significantly decreased miRNA expression of let-7a, miR-24, miR-34c, miR-100, miR-125b, and greatly increased miR-181a. The aberrant expression of miRNAs was associated with significantly increased protein expression of cyclins A, B1, D3 and E. Furthermore, remarkably enhanced expression of CDKs like CDK1, CDK2, CDK4, CDK6, especially p-CDK1 and p-CDK2 as well as alternations in the expression of pRB, p27, and CDC25A in the spleens of aniline-treated rats was also observed. The data suggest that aniline exposure leads to aberrant expression of miRNAs in the spleen which could be important in the regulation of cell cycle proteins. Our findings, thus, provide new insight into the role of miRNAs in cell cycle progression, which may contribute to aniline-induced tumorigenic response in the spleen.     

Epirubicin-mediated expression of miR-302b is involved in osteosarcoma apoptosis and cell cycle regulation

Epirubicin is widely used in osteosarcoma chemotherapy. Growing evidence indicates that the microRNA (miRNA) expression levels which are induced by chemotherapeutic agents play an important role in osteosarcoma development and progression. In this study we investigate the alterations of miRNA expression in the osteosarcoma cells after epirubicin treatment and whether miRNAs can enhance its anti-osteosarcoma effect. After epirubicin exposure, microarray shows 40 miRNAs are differentially expressed in osteosarcoma cells including 24 down-regulated miRNAs. Notably, miR-302b, which is stably low-expressed in osteosarcoma, could be induced by the epirubicin. Furthermore, we find that miR-302b can inhibit the osteosarcoma cell proliferation, promote cell apoptosis and cell cycle arrest MiR-302b can activate caspase-3 and regulate the Akt/pAkt, Bcl-2, Bim expression to increase the cell apoptosis. Meanwhile, miR-302b also attenuates cyclin D1 and CDKs expression to induce cell cycle arrest. Therefore, our results suggest miR-302b can play an essential role in osteosarcoma treatment as a potential tumor suppressor.     

Aberrant expression of miRNA-192-5p contributes to N,N-dimethylformamide-induced hepatic apoptosis

Excessive exposure to N,N-dimethylformamide (DMF) can lead to occupational liver poisoning in workers; however, the underlying mechanism is not fully clarified. The importance of microRNAs (miRNAs) in chemical-induced hepatotoxicity has been demonstrated. To determine whether miRNAs are also involved in DMF-induced hepatotoxicity, we systematically analyzed the miRNA expression profiles in DMF-treated (75 and 150 mm ) HL-7702 liver cells and controls by high-throughput sequencing. Among the altered miRNAs, miR-192-5p was the most significantly upregulated in HL-7702 cells after DMF exposure and was involved in DMF-mediated cell apoptosis. By contrast, suppression of miR-192-5p in HL-7702 cells attenuated the apoptosis induced by DMF. Furthermore, the anti-apoptotic gene (NIN1/RPN12 binding protein 1 homolog [NOB1]) was predicted to be a potential miR-192-5p target according to bioinformatics analysis. The direct interaction between miR-192-5p and NOB1 was confirmed by the dual-luciferase activity assay in HEK293FT cells. Overexpression of miR-192-5p efficiently reduced NOB1 mRNA and protein expression in HL-7702 cells. Alteration in NOB1 expression influenced DMF-induced hepatotoxicity by affecting hepatic apoptosis. In addition, the inverse correlation between miR-192-5p expression levels and NOB1 expression was further confirmed in DMF-exposed mouse liver tissue samples. These observations demonstrated that promotion of apoptosis from the suppression of NOB1 by miR-192-5p overexpression was responsible for the DMF-induced hepatotoxicity. This work provides the molecular mechanism at the miRNA level for hepatic apoptosis induced by DMF.     

MiR-320 and miR-494 affect cell cycles of primary murine bronchial epithelial cells exposed to benzo[a]pyrene

MicroRNAs (miRNAs) are a class of small noncoding RNA molecules with profound impact on various biological processes. Some miRNAs are involved in tumorigenesis by regulation of cell cycle progression. Here, we cultured primary murine bronchial epithelial cells and then examined the expression of miR-320 and miR-494 in cells exposed to benzo[a]pyrene (B[a]P). To better characterize roles of miR-320 and miR-494 in cell cycle progression, we used miRNA inhibitors to downregulate expression of miRNAs and determined cell cycle distribution and expression of cyclin-dependent kinases 6 (CDK6) by flow cytometric analysis. Treating cells with 1 μM B[a]P for 24 h resulted in time-dependent increases in miR-320 and miR-494 expression. Moreover, G1 arrest and downregulated expression of CDK6 were shown in the treated cells. Flow cytometric analysis indicated a relief of G1 arrest and an elevated expression of CDK6 after inhibition of the expressions of miR-320 and miR-494 in cells exposed to B[a]P. These results suggest that expression levels of miRNA-320 and miR-494, which regulate B[a]P-exposed cell cycle progression, may impact G1/S transition through CDK6, and provide further insights into functions of miRNAs in cell cycle of primary murine bronchial epithelial cells exposed to B[a]P.     

Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS

Perfluorooctanesulfonate (PFOS) is a persistent organic pollutant, the potential toxicity of which is causing great concern. In the present study, we employed zebrafish embryos to investigate the developmental toxicity of this compound. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to 0.1, 0.5, 1, 3 and 5 mg/L PFOS. Hatching was delayed and hatching rates as well as larval survivorship were significantly reduced after the embryos were exposed to 1, 3 and 5 mg/L PFOS until 132 hpf. The fry displayed gross developmental malformations, including epiboly deformities, hypopigmentation, yolk sac edema, tail and heart malformations and spinal curvature upon exposure to PFOS concentrations of 1 mg/L or greater. Growth (body length) was significantly reduced in the 3 and 5 mg/L PFOS-treated groups. To test whether developmental malformation was mediated via apoptosis, flow cytometry analysis of DNA content, acridine orange staining and TUNEL assay was used. These techniques indicated that more apoptotic cells were present in the PFOS-treated embryos than in the control embryos. Certain genes related to cell apoptosis, p53 and Bax, were both significantly up-regulated upon exposure to all the concentrations tested. In addition, we investigated the effects of PFOS on marker genes related to early thyroid development (hhex and pax8) and genes regulating the balance of androgens and estrogens (cyp19a and cyp19b). For thyroid development, the expression of hhex was significantly up-regulated at all concentrations tested, whereas pax8 expression was significantly up-regulated only upon exposure to lower concentrations of PFOS (0.1, 0.5, 1 mg/L). The expression of cyp19a and of cyp19b was significantly down-regulated at all exposure concentrations. The overall results indicated that zebrafish embryos constitute a reliable model for testing the developmental toxicity of PFOS, and the gene expression patterns in the embryos were able to reveal some potential mechanisms of developmental toxicity.     

Effects of short-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on microRNA expression in zebrafish embryos

Although many drugs and environmental chemicals are teratogenic, the mechanisms by which most toxicants disrupt embryonic development are not well understood. MicroRNAs, single-stranded RNA molecules of ~ 22 nt that regulate protein expression by inhibiting mRNA translation and promoting mRNA sequestration or degradation, are important regulators of a variety of cellular processes including embryonic development and cellular differentiation. Recent studies have demonstrated that exposure to xenobiotics can alter microRNA expression and contribute to the mechanisms by which environmental chemicals disrupt embryonic development. In this study we tested the hypothesis that developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known teratogen, alters microRNA expression during zebrafish development. We exposed zebrafish embryos to DMSO (0.1%) or TCDD (5 nM) for 1 h at 30 hours post fertilization (hpf) and measured microRNA expression using several methods at 36 and 60 hpf. TCDD caused strong induction of CYP1A at 36 hpf (62-fold) and 60 hpf (135-fold) as determined by real-time RT-PCR, verifying the effectiveness of the exposure. MicroRNA expression profiles were determined using microarrays (Agilent and Exiqon), next-generation sequencing (SOLiD), and real-time RT-PCR. The two microarray platforms yielded results that were similar but not identical; both showed significant changes in expression of miR-451, 23a, 23b, 24 and 27e at 60 hpf. Multiple analyses were performed on the SOLiD sequences yielding a total of 16 microRNAs as differentially expressed by TCDD in zebrafish embryos. However, miR-27e was the only microRNA to be identified as differentially expressed by all three methods (both microarrays, SOLiD sequencing, and real-time RT-PCR). These results suggest that TCDD exposure causes modest changes in expression of microRNAs, including some (miR-451, 23a, 23b, 24 and 27e) that are critical for hematopoiesis and cardiovascular development.     

Circulating microRNA profiles altered in mice after 28 d exposure to perfluorooctanoic acid

Perfluorooctanoic acid (PFOA) is a stable man-made compound with many industrial and commercial uses. Recently, however, concern has been raised that it may induce various toxicological effects such as hepatotoxicity, immunotoxicity, and developmental toxicity. Because levels of circulating microRNAs (miRNAs) can be altered in several clinical diseases, they may serve as potential novel biomarkers. Here, we explored differences in the profiles of circulating miRNAs in mice after PFOA exposure. Using TaqMan miRNA arrays, we determined that the levels of 24 circulating miRNAs were altered in mice dosed with PFOA at 1.25 mg/kg/d and 73 were altered in mice dosed with 5 mg/kg/d. Eight miRNAs were further validated using TaqMan Real-Time PCR assays. Results were consistent with those obtained from the TaqMan miRNA arrays, except for miR-199a-3p. The most remarkable of the circulating miRNAs (miR-26b-5p and miR-199a-3p) were also up-regulated in the serum of occupational workers in our previous epidemiological study. We also found similar patterns in mice exposed to PFOS. These results demonstrated that circulating miRNA profiles were altered after exposure to high concentrations of PFOA and miR-28-5p, miR-32-5p, miR-122-5p, miR-192-5p, and miR-26b-5p in serum may be linked to effects of PFOA, especially in occupationally exposed people.     

MicroRNA expression profiling involved in MC-LR-induced hepatotoxicity using high-throughput sequencing analysis

Microcystin-LR (MC-LR), the most common microcystin (MC) present in water is known to pose a significant threat to human health especially hepatotoxicity. However, the specific molecular mechanisms underlying MC-LR-induced hepatic cellular damage still remain to be determined. MicroRNAs (miRNAs) are known to play key roles in cellular processes including development, cell proliferation and responsiveness to stress. Thus, this study aimed to examine, whether miRNAs were involved in the observed MC-LR-mediated liver damage using miRNA profiling of a human normal liver cell line HL7702 using high-throughput sequencing techniques. Protein phosphatase 2A (PP2A) activity, an established biomarker of microcystin toxicity, was determined 24 hr following treatment with the algal toxin to confirm responsiveness. Data demonstrated that MC-LR significantly inhibited PP2A activity in a concentration-dependent manner with inhibitory concentration (IC₅₀) value of 4.6 μM. Compared with control cells, treatment with MC-LR at concentrations of 1, 2.5, 5 or 10 μM significantly modified expression of levels of 3, 10, 9, and 99 miRNAs, respectively. Expression levels of miR-15b-3p were significantly increased in all 4 treatment groups, while miR-4521 expression levels were markedly reduced. In the case of miR-451a, 1, 5 or 10 μM also significantly lowered expression levels. However, a significant rise in miR-451a was noted in cells exposed to 2.5 μM toxin. The results obtained from miRNA differential expression levels were confirmed by real-time fluorescent quantitative PCR (qPCR). Gene Ontology (GO) enrichment analysis of hepatic cells demonstrated that miRNAs significantly altered were involved in systems development, metabolism, and protein binding. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis data showed that target genes of differentially expressed miRNAs in liver cells predominantly participated in mechanistic target of rapamycin kinase (mTOR), Ras, Ras-related protein 1 (Rap1), hypoxia inducible factor 1 (HIF-1), and cancer development. In summary, evidence indicates that MC-LR-induced hepatotoxicity may be associated with alterations in miRNAs. Evidence indicates that alterations in miR-451a, miR-4521 and miR-15b-3p may be involved in the observed MC-LR- induced hepatotoxicity     

Cytoarchitectonic and neurochemical differentiation of the visual system in ethanol-induced cyclopic zebrafish larvae

Embryonic exposure to ethanol leads to malformations such as cyclopia. Cyclopic embryos present fused eyes and lack of the ventral specification of the brain, with physiological and morphological defects in the visual system, which provides a useful model for teratology and neurotoxicity assessments.We analysed the differentiation of the visual areas in the ethanol-induced cyclopic animals. For this purpose we exposed zebrafish embryos to 1.5% ethanol from 4 hours post-fertilisation (hpf) to 24 hpf in order to get cyclopic embryos. We monitored cytoarchitecture and quantified both the proliferation rate and cell differentiation from 2 days post-fertilisation (dpf) onwards, focusing on the main components of the visual system (retina, optic nerve and optic tectum) of normal and cyclopic zebrafish embryos.The visual system of the zebrafish embryos is affected by exposure to ethanol; two optic nerves that fuse before leaving the eyes are present in cyclopic specimens but an optic chiasm is not evident. Cell differentiation is severely delayed throughout the visual system at 2 dpf. At 5 dpf, lamination in the cyclopic retina and optic tectum is completed, but they are filled with pyknotic nuclei demonstrating cell death. At this stage the proliferation rate and expression patterns are unaltered and glial and neuronal neurochemical differentiations are similar to untreated animals. We found that the alterations produced by exposure to ethanol are not only cell-selective, but also tissue-selective.Cyclopia is the most severe phenotype induced by ethanol, although cell differentiation and proliferation can reach normal patterns after a certain period of time, which points to a neural plasticity process. Zebrafish embryos may possess a compensation mechanism against the ethanol effect, which would account for their use for pharmacogenetic and chemical screenings in the analysis of new molecules that could improve visual problems.     

The changes of miRNA expression in rat hippocampus following chronic lead exposure

miRNAs have been found to contribute to normal brain functions, nervous system diseases, as well as neurotoxicities induced by external agents. However, whether they are involved in lead-induced neurotoxicities is still not clear. To identify that, a lead-induced chronic neurotoxicity model of rats was built. Both miRNA microarray analysis and qRT-PCR were performed to determine the change of miRNA expression in hippocampus. Then 3 bioinformatics databases were used to analyze the relative target genes of these miRNA, which were further confirmed by qRT-PCR and Western blot. In the present study, lead exposure resulted in the changed expression of 7 miRNAs: miR-204, miR-211, miR-448, miR-449a, miR-34b, and miR-34c were greatly up-regulated while miR-494 was greatly down-regulated. Bioinformatics analysis results showed that the target genes of 6 up-regulated miRNAs were related to neural injury and neurodegeration, axon and synapse function, neural development and regeneration. Correspondingly, the expression levels of mature mRNAs and proteins of three target genes (Bcl-2, Itpr1, and Map2k1) were greatly repressed, verifying the results of bioinformatics analysis. Taken together, our results showed that the expression of several miRNAs reported to be associated with neurophysiological pathways and neurodegenerative diseases changed in rat hippocampus following chronic lead exposure. These miRNAs may play important roles in lead-induced neurotoxicity.     

MicroRNA-21表达调控研究

MicroRNAs(miRNAs)是一类广泛存在于真核细胞中的长约22 nt的非蛋白编码的、单链RNA,具有转录后基因调控的功能,与细胞分化、生长以及代谢等多种生物学过程的调节有关。研究发现,人体很多肿瘤中都存在miRNA的差异表达。而在这些差异表达的miRNA中,又以miR-21(miRNA-21)较为特殊。因为miR-21在几乎所有实体肿瘤中都存在高表达,而且高表达的miR-21可能发挥类似原癌基因的作用。miR-21通过负调节靶基因增加肿瘤细胞的增殖、凋亡以及肿瘤侵入转移,这些靶基因有PTEN、PDCD4、RECK等等。该文主要介绍miR-21在肿瘤中的作用、调节机制,以及作为靶基因在肿瘤的治疗中的临床意义。     

Abnormal development of motor neurons in perfluorooctane sulphonate exposed zebrafish embryos

Perfluorooctane sulfonate (PFOS) is an environmental organic pollutant, the potential neurotoxicity of which is causing great concern in fish. In the present study, we examined the effects of PFOS on motor neurons, and investigated the potential toxicological mechanisms oxidative stress in zebrafish embryos. Six-hour post-fertilization (hpf) zebrafish embryos were exposed to 1.0 mg/L PFOS, then we examined the expression of alpha-tubulin, proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase 5 (CDK5), and peroxiredoxin 2 (PRX2) after PFOS exposure until 120 hpf. The results showed that PFOS increased alpha-tubulin in the coccygeal spinal cord (CSC) at 96 hpf, whereas decreased alpha-tubulin in the brain and spinal cord at 120 hpf. PCNA expression was highly increased in CSC and abdomen compared with control at 96 and 120 hpf after PFOS exposure. In addition, PFOS exposure caused CDK5 expression to be highly increased in brain region following by down-regulation of PRX2 expression at 96 hpf. These results indicated that, at least in part, the effect on motor neurons induced by PFOS was mediated by dynamically interfering with the expression of alpha-tubulin and PCNA. Furthermore, PFOS-induced toxicity was associated with oxidative stress by deregulating CDK5 and PRX2.