Moon-Ju Oh

miRNA regulation of cytotoxic effects in mouse Sertoli cells exposed to nonylphenol.

BackgroundIt is known that some environmental chemicals affect the human endocrine system. The harmful effects of endocrine disrupting chemical (EDC) nonylphenol (NP) have been studied since the 1980s. It is known that NP adversely affects physiological functions by mimicking the natural hormone 17 beta-estradiol. In the present study, we analyzed the expression of miRNAs and their target genes in mouse Sertoli TM4 cells to better understand the regulatory roles of miRNAs on Sertoli cells after NP exposure.MethodsMouse TM4 Sertoli cells were treated with NP for 3 or 24 h, and global gene and miRNA expression were analyzed using Agilent mouse whole genome and mouse miRNA v13 arrays.ResultsWe identified genes that were > 2-fold differentially expressed in NP-treated cells and control cells (P < 0.05) and analyzed their functions through Gene Ontology analysis. We also identified miRNAs that were differentially expressed in NP-treated and control cells. Of the 186 miRNAs the expression of which differed between NP-treated and control cells, 59 and 147 miRNAs exhibited 1.3-fold increased or decreased expression at 3 and 24 h, respectively. Network analysis of deregulated miRNAs suggested that Ppara may regulate the expression of certain miRNAs, including miR-378, miR-125a-3p miR-20a, miR-203, and miR-101a, after exposure to NP. Additionally, comprehensive analysis of predicted target genes for miRNAs showed that the expression of genes with roles in cell proliferation, the cell cycle, and cell death were regulated by miRNA in NP-treated TM4 cells. Levels of expression of the miRNAs miR-135a* and miR-199a-5p were validated by qRT-PCR. Finally, miR-135a* target gene analysis suggests that the generation of reactive oxygen species (ROS) following exposure to NP exposure may be mediated by miR-135a* through regulation of the Wnt/beta-catenin signaling pathway.ConclusionsCollectively, these data help to determine NPs actions on mouse TM4 Sertoli cells and increase our understanding of the molecular mechanisms underlying the adverse effects of xenoestrogens on the reproductive system.

Impact of miRNA deregulation on mRNA expression profiles in response to environmental toxicant, nonylphenol

MicroRNA (miRNA) are approximately 22nt RNA molecules with the ability to regulate gene expression by interacting with its target mRNA. Recent studies demonstrated that miRNA are responsible for determining cell fate and also plays an important role in cellular response to xenobiotics stress and other toxicological phenomenon. Also a number of reports have strengthened the correlation between altered miRNA expression and various cancers. In this study miRNA and mRNA expression profiling was carried out in in-vitro differentiating MCF-7 and HepG2 cell line to understand the effect of nonylphenol (NP), an industrially synthesized environmental toxicant. Analyzing the mRNA-miRNA interaction, we observed correlation between deregulated miRNAs and its predictive differentially expressed target mRNA. We also observed differential expression of two widely studied miRNAs, miR-21 and miR-34 in these cell lines. CTCF is found to be the predictive target of expressed miR-21 in MCF-7. In HepG2 cell line expressed MDM2 is found to be a predictive target of miR-34b. These results support the possible role of miRNA interaction in the expression of its target genes and also ability of environmental toxicant to deregulate miRNA expression.

Gene expression profiling of HepG2 cells treated with endocrine disrupting chemicals using the HazChem human array V3

The endocrine system is a system of glands that secrete hormones to regulate the body. This system is disrupted by endocrine disrupting chemicals (EDCs), which are similar to sex hormones. These chemicals function as androgen antagonists or estrogen agonists. To determine the effect of EDCs on the gene expression profile in human cells, we treated HepG2 cells with 3 chemicals (bisphenol A, 17β-estradiol, vinclozolin), and analyzed common gene expression using a custom-made HazChem human array V3. The Haz-Chem human array V3 included a total of 1136 genes, all of which were differentially expressed by exposure to VOCs, PAHs, POPs, and LTCs. Of these genes, 24 genes were commonly expressed after exposure to all 3 chemicals, based on the SAM (Significant Analysis of Microarray) method (q-value < 0.5%). These genes were analyzed further and found to be involved in coagulation, hemostasis, wound healing, angiogenesis, homeostasis, cell redox homeostasis, and cell proliferation based on GO function and pathway networks.

Genomic comparison of insecticides and herbicide in human hepatoma (HepG2) cell line

Pesticide is a chemical substance, biological agent, antimicrobial, disinfectant or device used against any pest, and it can be grouped in several different ways. Atrazine is one of the most commonly used herbicides found in the rural environments, permethrin and prallethrin are pyrethroid insecticides. In this study, we compared the characters of herbicide and insecticide by cytotoxicity and microarray experiments. In the cytotoxicity tests, we couldn’t find specific chemical features. However, in the gene expression analysis, we found that insecticides affects to RNA processing and RNA metabolism, and especially, in the energy metabolism, such as generation of precursor metabolites and energy, and energy derivation by oxidation of organic compounds, herbicides and insecticides are differently working. This study provides characteristics of two pesticides by checking the transcriptional change.

Analysis of microRNA and gene expression profiling in triazole fungicide-treated HepG2 cell line.

MicroRNA (miRNA) plays an important role in various diseases and in cellular and molecular responses to toxicants. In the present study, we investigated differential expression of miRNAs in response to three triazole fungicides (myclobutanil, propiconazole, and triadimefon). The human hepatoma cell line (HepG2) was treated with the above triazoles for 3 h or 48 h. miRNA-based microarray experiments were carried out using the Agilent human miRNA v13 array. At early exposure (3h), six miRNAs were differentially expressed and at late exposure (48 h), three miRNAs were significantly expressed. Overall, this study provides an array of potential biomarkers for the above triazole fungicides. Furthermore, these miRNAs induced by triazoles could be the foundation for the development of a miRNA-based toxic biomarker library that can predict environmental toxicity.

The study of diethylstilbestrol toxic effect in the mouse sertoli cell line by comparison of miRNA and mRNA expression

It is important to acknowledge the harmful effects of environmental chemicals in human’s lives. The toxic effects of Diethylstilbestrol (DES), one of the endocrine-disrupting chemicals (EDCs), have been documented in many studies. As expected, DES affect male gendal hormone as well as female’s; therefore, epigenetic study should be considered. In this study, microarray technology was used to study harmful effects on the level of genomics, and here, two types of microarray chips- the Agilent mouse genome 4 × 44 K array for gene expression profiling and the Agilent mouse miRNA v13 for miRNA expression profiling-was used to study the relation between gene and miRNA expression profiles. As a result, we identified 4 miRNAs (miR 203, 350, 421, and 466i) that were similarly expressed at 3 hrs and 24 hrs of DES treat times. Twenty one genes matched between predicted target for 4 miRNAs and 118 genes expressed similarly. These genes have functions related to cell differentiation and cell cycle. Therefore, DES affects cellular function and induces toxicity in TM4 cells. In future studies, it is necessary to find more related functions and mechanisms of DES in the system.

Identification of time-dependent biomarkers by EndoTox Array in cells exposed to nonylphenol

Nonylphenol (NP) is considered an endocrine disruptor due to its weak ability to mimic estrogen and in turn disrupt the natural balance of hormones in affected organisms. NP is reported to cause negative health effects in humans, such as hormone abnormalities and inhibition of growth and reproduction. In the present study, we developed a molecular tool for the evaluation of endocrine toxicity in mouse. To identify gene regulation effects of NP, we estimated gene expression in mouse Sertoli (TM4) and germ cell (GC) lines after exposure to NP. We measured the IC30 value of NP, then exposed the cells to that concentration for 3 hr and 24 hr and used EndoTox Array. The EndoTox Array was manufactured to monitor the endotoxicity of environmental chemicals. This array contains 1306 genes that are influenced in reproductive toxicity or by EDCs. In the expression pattern analysis, 28 genes related to the reproductive process, cell proliferation, and nervous system development were progressively changed over time in NP-exposed cells. The study of gene interaction will increase our understanding of the time-dependent molecular mechanisms of NP.

A new approach of digital PCR system for non-invasive prenatal screening of trisomy 21

BACKGROUND Non-invasive prenatal screening (NIPS) of trisomy 21 (T21) using digital PCR (dPCR) with several advantages will be very effective. Here, we developed a dPCR system for T21 screening which allows high sensitivity and real-time diagnosis and thus overcome sequence based analysis. METHODS Cut-off value was established using DNA extracted from all 157 T21 negative samples including 47 pregnant woman samples and 3 T21 positive pregnant woman samples extracted from 4 different sample types. To increase the portion of the cell-free fetal DNA (cffDNA) in maternal cell-free DNA (cfDNA), a size selection method was devised. We evaluated the clinical reliability of NIPS using dPCR through analysis of 877 pregnant woman samples. RESULTS We could demonstrate the possibility of NIPS using dPCR performed by applying cut-off value and size selection method. The overall accuracy was derived at 99.66% using 877 pregnant woman plasma samples. CONCLUSION Our results showed that dPCR can meet the requirements for NIPS for T21. It is relatively inexpensive, easy to use in a screening method and compatible with ethical concerns regarding access to nucleotide sequence information. This study may be a basic data for the development of population-wide screening for T21 in pregnant women.