Prenatal Exposure to TCDD Triggers Significant Modulation of microRNA Expression Profile in the Thymus That Affects Consequent Gene Expression

Abstract

BackgroundMicroRNAs (miRs) are a class of small RNAs that regulate gene expression. There are over 700 miRs encoded in the mouse genome and modulate most of the cellular pathways and functions by controlling gene expression. However, there is not much known about the pathophysiological role of miRs. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), an environmental contaminant is well known to induce severe toxicity (acute and chronic) with long-term effects. Also, in utero exposure of fetus to TCDD has been shown to cause thymic atrophy and alterations in T cell differentiation. It is also relevant to understand “the fetal basis of adult disease” hypothesis, which proposes that prenatal exposure to certain forms of nutritional and environmental stress can cause increased susceptibility to clinical disorders later in life. In the current study, therefore, we investigated the effects of prenatal exposure to TCDD on miR profile in fetal thymocytes and searched for their possible role in causing thymic atrophy and alterations in the expression of apoptotic genes.Methodology/Principal FindingsmiR arrays of fetal thymocytes post exposure to TCDD and vehicle were performed. Of the 608 mouse miRs screened, 78 miRs were altered more than 1.5 fold and 28 miRs were changed more than 2 fold in fetal thymocytes post-TCDD exposure when compared to vehicle controls. We validated the expression of several of the miRs using RT-PCR. Furthermore, several of the miRs that were downregulated contained highly complementary sequence to the 3′-UTR region of AhR, CYP1A1, Fas and FasL. Also, the Ingenuity Pathway Analysis software and database was used to analyze the 78 miRs that exhibited significant expression changes and revealed that as many as 15 pathways may be affected.Conclusions/SignificanceThese studies revealed that TCDD-mediated alterations in miR expression may be involved in the regulation of its toxicity including cancer, hepatic injury, apoptosis, and cellular development.

Exposure to Diethylstilbestrol during Pregnancy Modulates MicroRNA Expression Profile in Mothers and Fetuses Reflecting Oncogenic and Immunological Changes

“…The data generated from miR arrays were analyzed using a hierarchical clustering and pathway network. Induction or repression of miR expression was determined using a twosample t test as described previously (Singh et al, 2012a;Hegde et al, 2013), and a P value of ,0.01 in the t test was considered significant. In this study, a fold change of more than 1.5-fold was considered positive.…”

“…Postgeneration of the heat map and analysis of miR expression, we selected dysregulated (upregulated/downregulated) miRs (.1.5 fold) in fetal thymi exposed to DES when compared with those exposed to VEH. Next, the selected miRs were analyzed for their role in the expression of various genes and pathways using IPA software and the database of the company (Ingenuity Inc., Redwood City, CA) as described previously (Singh et al, 2012a;Hegde et al, 2013). DESregulated miRs were also analyzed using Cytoscape 3.0.1 and Cluego software (Cytoscape Consortium, San Diego, CA) as described previously (Singh et al, 2012a;Hegde et al, 2013).…”

“…We identified miR-specific mRNA targets using microRNA.org (Memorial Sloan Kettering Cancer Center, New York, NY), TargetScan mouse 5.2 (Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Boston, MA), miRWalk (Heidelberg, Germany), and miRGEN (version 3; Diana Lab, Athens, Greece) software and their database as described earlier (Singh et al, 2012a;Hegde et al, 2013). The details of some of miRs and the UTR region of their target gene (RNA targets) are described in Supplemental Table 2.…”

“…EL4 cells (5 Â 10 6 ) were transfected using the lipofectamine RNAMAX transfection kit from Invitrogen and following the protocol (reverse transfection) of the company (Life Technologies) as described previously (Singh et al, 2012a;Hegde et al, 2013). Forty-eight hours post-transfection, EL4 cells were treated with VEH or DES (10 mM/ml) for 24 hours, and the expression of Fas and FasL in EL4 cells was determined.…”

“…To this end, total RNAs from EL4 cells transfected with miR-23a or miR-18b and treated with DES or VEH were isolated using the RNeasy mini kit from QIAGEN and following the protocol of the company. Fas and FasL expression was determined as described previously (Singh et al, 2012a). The PCR products generated from mouse Fas and FasL primer pairs were normalized against PCR products generated from mouse 18S after electrophoresis.…”