Exposure to diethylstilbestrol during pregnancy modulates microRNA expression profile in mothers and fetuses reflecting oncogenic and immunological changes
2015 Study 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 3′ 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.
DES Induces Alterations in Thymic Cellularity in Mothers and Prenatal Fetuses
We have previously reported that prenatal exposure to DES induces thymic atrophy and apoptosis resulting from upregulation of Fas and Fas ligand expression :
- Induction of apoptosis in murine fetal thymocytes following perinatal exposure to diethylstilbestrol.
- Prenatal exposure of mice to diethylstilbestrol disrupts T-cell differentiation by regulating Fas/Fas ligand expression through estrogen receptor element and nuclear factor-κB motifs.
In this study, our goal was to investigate the role of microRNA in such immunologic changes induced by DES.
To this end, we administered a single dose of DES (5 μg/kg) into pregnant C57BL/6 mice on GD 14 as described previously and studied the effect on the thymocytes. On days 2 and 3 post-DES treatment (GD 16 and GD 17), the thymic cellularity of mothers and fetuses was determined. There was a significant decrease in the thymic cellularity of the mothers on both day 2 (GD 16) and day 3 (GD 17) post-DES exposure. In fetuses, however, no significant difference in thymic cellularity was observed on day 2 (GD 16), but a significant decrease in thymic cellularity of fetuses was observed on day 3 (GD 17) post-DES exposure. These data are consistent with previously published results from our laboratory that showed DES induces a decrease in thymic cellularity in both pregnant mothers and fetuses. We also confirmed in these experiments that exposure to DES led to increased apoptosis in thymocytes from both mothers and fetuses and that DES-exposed cells had higher levels of Fas and FasL expression (data not shown), which is consistent with our earlier studies.
Analysis of DES-Regulated miR Profile in Thymocytes from the Mother and Fetus
The data obtained from miR arrays of the thymocytes post-DES exposure were further analyzed. To this end, cluster analyses of 609 miRs were performed using Ward’s method. Similarly, the measure of the miRs of the four groups [mother + VEH, mother + DES, fetus + VEH, and fetus + DES] was done using the half-square Euclidean distance method and ordering the function of miRs was done on the basis of input rank. Visualization of the cluster analysis of miRs has been presented as a dendrogram and their expression pattern is reflected. Upon comparison of more than 1-fold dysregulated (upregulated or downregulated) miRs in DES-treated mothers versus VEH-treated mothers or DES-treated fetuses versus VEH-treated fetuses, there were 59 miRs that were common for both mothers and fetuses, whereas there were 107 miRs that were specific to mothers only and 101 miRs that were specific to fetuses only.
Analysis of miRs Associated with Fas and FasL Expression
Previous studies from our laboratory have reported that DES induces an increased expression of Fas and FasL in thymocytes, which plays a critical role in the induction of apoptosis.
- Diethylstilbestrol alters positive and negative selection of T cells in the thymus and modulates T-cell repertoire in the periphery.
In this context, we investigated potential miRs involved in the expression of Fas and FasL. We used TargetScanMouse 5.2, miRWalk, and microRNA.org software, and detected a significant binding affinity of miR-23a and miR-18b with the 3′UTR region of Fas and FasL genes, respectively.
To confirm this analysis, we used EL4 T cells not transfected or transfected with mature miR-23a or miR-18b and cultured them in the absence or presence of DES for 24 hours. The expression of Fas and FasL was determined by performing RT-PCR. EL4 cells spontaneously expressed Fas and FasL (VEH treated), and treatment with DES further augmented their expression. Interestingly, expression of Fas in EL4 cells transfected with miR-23a was decreased significantly when compared with VEH-treated EL4 cells. Moreover, EL4 cells transfected with miR-23a and treated with DES also showed a decrease in Fas expression when compared with DES-treated cells. Similarly, upregulated expression of FasL was observed in EL4 cells treated with DES when compared with VEH-treated cells. Upon transfection of EL4 cells with miR-18b, there was significant downregulation of FasL expression, but upon DES treatment, downregulation of FasL expression was significantly reversed. Overall, in these studies, we also noted that transfection of vehicle-treated EL4 cells with miR-23a decreased the expression of only Fas but not FasL, and similarly, transfection with miR-18b decreased the expression of FasL but not Fas. Also, DES reversed the miR transfection-induced inhibition of the expression of Fas and FasL. These data together suggested that DES-induced decrease in the expression of miR-23a and miR-18b may indeed be the mechanism through which Fas and FasL, respectively, get upregulated.
In summary, we demonstrate for the first time that prenatal exposure to DES can cause a significant effect on the miR profile in the thymuses of both the mother and fetus. These upregulated or downregulated miRs may influence the regulation of genes that affect the development of the immune cells and other organ systems. Identification of miRs as targets for DES-induced modulation of gene expression offers novel mechanisms to understand DES-regulated molecular mechanisms and its long-term effects.
Sources and more information
- Full text (free access) : Exposure to Diethylstilbestrol during Pregnancy Modulates MicroRNA Expression Profile in Mothers and Fetuses Reflecting Oncogenic and Immunological Changes, Molecular pharmacology, NCBI PubMed PMC4407731, 2015 May.
- Heat map of miR expression profile in thymi of mothers and fetuses postexposure to DES featured image credit PMC4407731/figure/F2.