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

Abstract

Prenatal exposure to diethylstilbestrol (DES) is known to cause an increased susceptibility to a wide array of clinical disorders in humans. Previous studies from our laboratory demonstrated that prenatal exposure to DES induces thymic atrophy and apoptosis in the thymus. In the current study, we investigated if such effects on the thymus result from alterations in the expression of microRNA (miR). To that end, pregnant C57BL/6 mice who were exposed to DES and miR profiles in thymocytes of both the mother and fetuses on postnatal day 3 (gestation day 17) were studied. Of the 609 mouse miRs examined, we noted 59 altered miRs that were common for both mothers and fetuses, whereas 107 altered miRs were specific to mothers only and 101 altered miRs were specific to fetuses only. Upon further analyses in the fetuses, we observed that DES-mediated changes in miR expression may regulate genes involved in important functions, such as apoptosis, autophagy, toxicity, and cancer. Of the miRs that showed decreased expression following DES treatment, miR-18b and miR-23a were found to possess complementary sequences and binding affinity for 39 untranslated regions of the Fas ligand (FasL) and Fas, respectively. Transfection studies confirmed that DES-mediated downregulation of miR-18b and miR-23a led to increased FasL and Fas expression. These data demonstrated that prenatal DES exposure can cause alterations in miRs, leading to changes in the gene expression, specifically, miR-mediated increased expression in FasL and Fas causing apoptosis and thymic atrophy.

Dietary Indoles Suppress Delayed-Type Hypersensitivity by Inducing a Switch from Proinflammatory Th17 Cells to Anti-Inflammatory Regulatory T Cells through Regulation of MicroRNA

“…In brief, total RNA including miRs from inguinal LNs were isolated using miRNeasy kit and following the protocol of the company (QIAGEN, Valencia, CA). Next, miR arrays were performed and data were analyzed using hierarchical clustering, as described previously (33, 44, 55). miR arrays data were submitted to Array Express (Accession number: E-MTAB-4076; webpage: http://www.ebi.ac.uk/arrayexpress/submit/overview.html; https://www.ebi.ac.uk/fg/annotare/).…”

“…The selection of these two miRs was also based on their binding affinity with complementary sequences of their respective genes (miR-490:FoxP3 and miR-1192: IL-17). The transfections were pursued as described earlier (33, 44, 55, 56). Briefly, T cells purified from LNs of mBSA-immunized mice were either transfected with scrambled miR (MOCK) or with mature miR-490 mimic, miR-490 inhibitor using Transfection kit from QIAGEN and following the protocol of the company (QIAGEN).…”

Oestrogenic Endocrine Disruptors in the Placenta and the Fetus

“…Due to exposure to DES may cause DNA methylation, the fetal genome is allowed to encode multiple defects, which may not be expressed before a certain period of time (puberty, for example) [95]. Studies have shown that microRNA levels may change due to the effects of DES, and microRNA-mediated upregulation of Fas and its ligands leads to increased autophagy and apoptosis [96].…”

Diethylstilbestrol (DES) induces autophagy in thymocytes by regulating Beclin-1 expression through epigenetic modulation

“…We purchased Diethylstilbestrol (DES) powder and Acridine Orange (AO) solution from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO). DES suspended in corn oil was used for in vivo studies and DES suspended in Dimethyl sulfoxide (DMSO) was used for in vitro studies, as described (Singh et al, 2012, 2015a,b). The culture medium (RPMI1640, Penicillin/ Streptomycin, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), L-glutamine, Fetal Bovine Serum (FBS), and PBS (Phosphate-buffered saline) were purchased from Invitrogen Life Technologies (Carlsbad, CA).…”