환경독성물질에 의한 mRNA와 miRNA 발현 영향 연구

Abstract

Environmental chemicals can cause a broad spectrum of effects, causing threat to wildlife and humans health. These chemicals may adversely affect the function and/ or structure of living organism depending upon the degree and route of exposure. Exposure to such chemicals may trigger altering gene expression pattern in cells and disrupt variety of cellular functions. Thus toxicogenomics has emerged as fields that study this complex interaction between cellular responses to toxic agent in the environment. Thioacetamide (TA) and Nonylphenol (NP) are such environmental toxicants that are reported to impose many adverse effects on animals and humans. In this thesis toxicogenomic study was attempted to provide information regarding the mechanism of cellular response to these toxicants and identifying some possible marker genes in this complicated cellular response. First, we investigated the effect of TA in WB-F344 rat liver epithelial (WB-F344) cell. TA is a potent hepatotoxicant known to affect liver metabolism, inhibit mRNA transport and induce immune suppression. We analyzed the transcriptional profiling of WB-F344 cells after treatment with TA at the low (1,000 ?M) and high (10,000 ?M) dose concentrations over five time points (1, 3, 6, 12 and 24 hrs). Some of the genes identified with TA treatment were significant at early time (1hr and 3hrs) points, while that of others were at late time (6hrs, 12hrs and 24hrs) points. Many of the differential expressed genes at early time point are related to cytotoxicity and mainly implicated in signal transduction, apoptosis and G-protein receptor pathways. This result suggests that early response of genes may directly correlate with the specific mechanism underlying TA-induced cytotoxicity. Second, we analyzed change in transcriptional profiles in MCF-7 (human breast cancer) cells and HepG2 (human hepatocellular liver carcinoma) cells with response to NP using microarray. NP is considered as an environmental hazardous chemical which is extensively used in industry, institution and household application and is bio-accumulative and resistance to biodegradation. NP is also known to have estrogen-like activity and act as an endocrine disrupting chemical. We treated NP at 12?M and 52?M to MCF-7 and HepG2 respectively at two time point, 3hrs and 48 hrs. In both the cell lines, with NP exposure, we observed differential expression of many genes related to cytotoxicity with function such as immune response, metabolism, induction of inflammation, apoptosis and signaling pathways. Differential expression of the genes involved in these pathways can be considered as a good marker gene for detecting NP toxicity with exposure in human cell lines and shows microarray technology as a useful tool for detecting such environmental toxicant. In the final part of the thesis, to understand the underlying cellular mechanism and possible role of microRNA (miRNA) in regulating the expression of these marker genes, analysis of miRNA expression profiles is carried out in response to NP at 3hrs and 48hrs using microarray approach. miRNAs are endogenous small non-coding RNAs that regulate gene expression at posttranscriptional level. This complex gene regulatory circuitry has revolutionized our understanding of cellular physiological and pathological processes. Differential expression of many miRNAs (miR-766, miR-187, miR-205, miR-16, miR-195 etc) was observed but the pattern of miRNA expressed in both the cell lines were varied showing its tissue specificity. Analyzing the mRNA and miRNA interaction, we observed some of these expressed marker genes are target of some of the deregulated miRNAs. We also observed differential expression of two widely studied miRNA, miR-21 and miR-34 in these cell lines. CTCF is found to be the predictive target of expressed miR-21in MCF-7. In HepG2 cell line expressed MDM2 is found to be a predictive target of miR-34b which mediated down-regulation of miR-34a by deactivation of p53. 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. In summary, this thesis represents predictive marker genes due to exposure and effect of environmental toxicant. It also shows the effect of toxicant in the expression profile of miRNA and in turn effect of this miRNA change on its target gene. Study of expression profiles of mRNA and miRNA interaction indicates the underlying possible involvement of mRNA and miRNA in overall cellular and molecular function in response to environmental toxicants. Lastly, toxicogenomics study with microarray can be considered as a useful approach in understanding mRNA and miRNA interaction.