Steroidal regulation of uterine miRNAs is associated with modulation of the miRNA biogenesis components Exportin-5 and Dicer1.

Within the uterus, the female sex steroids estrogen and progesterone play pivotal roles in the establishment of a suitable environment for embryo implantation and pregnancy. More specifically, these steroids regulate a multitude of cellular processes that include cell proliferation cell differentiation, regulation of vascular permeability, angiogenesis, and adenogenesis all of which are essential for proper development and function of the adult uterus [ 1 – 7 ]. In order to bring about these changes, estrogen and progesterone must appropriately modulate the expression of a variety of factors which includes growth factors, cytokines, extracellular matrix proteins, and adhesion molecules [ 1 – 7 ]. In recent years, microRNAs (miRNAs) have been identified as a novel class of small, non-coding RNAs which also regulate gene expression by affecting mRNA translation and/or stability [ 8 , 9 ]. miRNAs are transcribed from specific genes which reside between protein-coding genes, within introns of genes, within exons or which make up the entire intron [ 10 , 11 ]. miRNA biogenesis begins with the transcription of primary RNA transcript (pri-miRNA) which then is processed to a 70–100 nucleotide precursor miRNA (pre-miRNA) by a protein complex composed of Drosha (a class 2 RNAse III-type enzyme), DGCR8 (a RNA-binding protein also known as DiGeorge syndrome critical region 8), and p68/72 DEAD-box RNA helicase [ 12 ]. Pre-miRNAs are then exported from the nucleus to the cytoplasm through exportin-5 [ 13 ] where they are subsequently processed to the mature 19–23 nucleotide miRNA by Dicer [ 14 ]. Mature miRNAs form a RNA-induced silencing complex (miRISC) along with Dicer, TAR RNA-binding protein, and Argonaut proteins [ 15 ]. The miRISC then binds target mRNA and regulates their translation by either cleaving and degrading the target transcript, deadenylating and degrading the target transcript, inhibiting mRNA translation initiation, inhibiting mRNA translation elongation, or mRNA translational enhancement [ 8 , 9 ]. MicroRNAs (through tissue specific knockdown experiments) have been shown to play a role in the normal development of the lung [ 16 ], limbs [ 17 ], skeletal muscle [ 18 ], female germline [ 19 ], embryo [ 20 ], oviduct [ 21 – 23 ], and uterus [ 22 , 23 ]. Recently, post-transcriptional regulation and miRNAs have been proposed to play a role in embryo implantation [ 24 , 25 ] as well as in the human endometrium [ 26 , 27 ] and in the pathophysiology of the female disease endometriosis [ 26 , 28 – 30 ]. Collectively, these studies suggest that miRNAs play a pivotal role in the molecular regulation of multiple organ systems, including reproductive function and specifically uterine implantation and uterine pathophysiological conditions. Unfortunately, beyond these limited studies there is no information on the regulation of key miRNA biogenesis components in vivo. The objectives of the current study were to examine steroidal regulation of uterine Drosha, Dgcr8, Exportin-5 and Dicer1 expression and to demonstrate that regulation of these miRNA biogenesis components occurs through steroid-receptor mediated pathways.

Estrogen regulation of Dicer1, Drosha, Dgcr8 , and Exportin-5 transcript was assessed by qRT-PCR ( Fig. 1 ). Estrogen had a minimal affect on the expression of Dicer1 and Drosha as their levels of expression did not significantly change in response to estrogen treatment. In contrast, estrogen induced a modest (1.6-fold) but significant increase in Dgcr8 expression at 8 h post steroid administration with levels decreasing to basal values at 24 h. Of the four miRNA biogenesis pathway components assessed, Exportin-5 ( Xpo5 ) was the most highly regulated by estrogen. Estrogen significantly increased Exportin-5 expression at both 4 and 8 h post steroid administration with levels returning to 0 h values by 24 h post estrogen treatment. Progesterone also regulated the expression of specific components of the miRNA biogenesis pathway ( Fig. 2 ). Dicer1 expression was significantly increased by progesterone at 8 h post steroid administration, while Drosha and Dgcr8 expression was not affected by progesterone treatment. Exportin-5 expression was also markedly regulated by progesterone. Progesterone significantly increased Exportin-5 expression at 8 and 24 h post steroid administration. Administration of estrogen and progesterone in combination resulted in a significant increase in all four components of the miRNA biogenesis pathway ( Fig. 3 ). Dicer1 expression was significantly increased at both 4 and 8 h post steroid treatment while Drosha was increased at 8 h post treatment. Dgcr8 expression was also significantly increased at 8 h post steroid treatment similar to estrogen alone (see Fig. 1 ). Similar to regulation by either estrogen or progesterone alone, concurrent administration of both steroids significantly increased the expression of Exportin-5 . Estrogen and progesterone together induced a 2-fold increase in Exportin-5 expression compared to either steroid alone (6-fold maximal increase versus 3-fold increase; see Figs. 1 , 2 ) and this increase was maintained from 4 h post steroid administration through 24 h post steroid administration. In order to determine if estrogen regulation of Exportin-5 and Dgcr8 was mediated through the cognate estrogen nuclear receptor pathway, ICI 182,780 antagonistic studies were employed. As displayed in Figs. 4 and 5 , respectively, the estrogen-induced increase in both Exportin-5 ( Xpo5 ; Fig. 4a ) and Dgcr8 ( Fig. 5 ) was significantly reduced by pre-treatment with ICI followed by estrogen challenge. ICI alone had no significant effect on either Exportin-5 or Dgcr8 transcript expression. Assessment of protein expression for Exportin-5 by Western analysis revealed that estrogen regulation of protein levels was similar to that of Exportin-5 transcript expression ( Fig. 4b ). Specifically, estrogen significantly ( P < 0.05) induced Exportin-5 expression over vehicle controls approximately 2.2-fold and this induction by estrogen was blocked by pre-treatment with ICI. ICI alone had no effect on Exportin-5 protein expression. In order to determine if the progesterone induced expression of Dicer1 and Exportin-5 were mediated through the cognate progesterone nuclear receptor pathway, RU-486 progesterone receptor antagonist studies were conducted (Figs. 6 , 7 ). Pre-treatment with RU-486 blocked the progesterone induction of both Dicer1 ( Fig. 6 ) and Exportin-5 ( Fig. 7 ) while RU-486 alone had no significant affect on expression of either miRNA biogenesis component. Assessment of protein expression for Dicer1 and Exportin-5 by Western analysis revealed that progesterone regulation of their protein levels was similar to that for their transcript expression. Specifically, progesterone induced a modest but significant increase (approximately 1.5-fold; P < 0.05) in Dicer1 expression over vehicle controls and this progesterone induction was blocked by pre-treatment with RU-486 (RU; Fig. 6 ). RU-486 alone had no effect on Dicer1 protein expression ( Fig. 6 ). Similarly, progesterone induced a significant ( P < 0.05; 1.8-fold) increase in Exportin-5 protein expression which could be inhibited by pre-treatment with RU-486 ( Fig. 7 ). Similar to Dicer1 protein, RU-486 alone had no significant effect on Exportin-5 protein expression. In order to verify that these steroid-induced changes in Exportin-5 and Dicer1 were associated with changes in liberation of mature miRNAs, we assessed miR-451 expression. We selected miR-451 as we had previously demonstrated that miR-451 is induced in uterine tissue in response to estrogen treatment [ 31 ]. miR-451 expression was assessed by qRT-PCR and results are summarized in Fig. 8 . miR-451 expression was significantly increased by estrogen alone (peaking at 4 h post-estrogen administration), progesterone alone (peaking at 8 h post-progesterone administration), and by both steroids together (peaking at 8 h post steroid administration). These patterns of induction are consistent with that of the miRNA biogenesis components and their induction by these steroids (see Figs. 1 , 2 , 3 ). Steroidal modulation of miR-451 expression through the estrogen receptor and progesterone receptor pathways was confirmed by antagonist studies. Pre-treatment with ICI blocked the estrogen induction of miR-451 while pre-treatment with RU-486 blocked the progesterone-induced expression of miR-451 ( Fig. 9 ). Collectively, these studies demonstrate that the liberation of mature miR-451 expression coincides with the induction of the miRNA biogenesis components and that this induction occurs through the cognate estrogen and progesterone receptor pathways.

miRNAs are proposed to be involved in diverse developmental and pathological processes. It is predicted that 1–5% of genes encode for miRNAs, and they regulate the expression of as many as 30% of mRNAs [ 32 , 33 ]. Not surprising, miRNAs have been proposed to regulate stability of mRNAs within the uterus that play a role in normal physiological processes which include cell proliferation, cell differentiation, apoptosis, angiogenesis, and inflammation. In contrast, alterations in the normal pattern of expression of uterine miRNAs have been postulated to play a role in endometrial pathologies such as endometriosis [ 26 , 28 – 30 ] as well as in human endometrial cancer [ 26 , 34 – 36 ] and animal models of endometrial carcinoma [ 37 ]. Expression profiles for uterine miRNAs in normal and pathological conditions provide the initial basis for determining which miRNAs may actively impact the uterine genome. Within the uterus, miRNAs have been shown to be regulated by the ovarian steroids estrogen and progesterone. Previous study from our lab [ 31 ] demonstrated that estrogen both increases and decreases the expression of specific uterine miRNAs in vivo. The ability of estrogen to modulate uterine miRNAs has also been examined in vitro using isolated endometrial epithelial and stromal cell cultures. Pan and colleagues [ 28 ] have demonstrated that estrogen induces specific miRNAs as well as represses the expression of others and that this regulation is mediated through the estrogen receptor-dependent pathway. In the same study, progesterone was also shown to modulate endometrial miRNA expression through its cognate receptor pathway. While these studies demonstrate that both estrogen and progesterone modulate the expression of specific miRNAs and that this regulation occurs through their respective receptor-mediated pathways, the precise mechanism by which these steroids modulate miRNA expression remains poorly understood. Mature miRNAs are first generated through transcription from the genome in the form of primary transcripts (pri-miRNAs). Pri-miRNAs are then processed within the nucleus by DGCR8 and Drosha, exported from the nucleus by Exportin-5 and processed in the cytoplasm by Dicer1 to liberate the mature miRNAs which are capable of regulating mRNA transcript stability. It is postulated that estrogen and progesterone induce transcription of pri-miRNAs through steroid receptor–promoter interactions but little to no data on this possibility has been presented. Estrogen response elements have been identified in specific miRNA [ 38 ] and this group has proposed that direct binding of the estrogen receptor to the regulatory regions of the miRNAs lead to their estrogen-induced expression. Additional possibilities proposed by Bhat-Nakshatr and colleagues include estrogen-inducible expression of mRNA-encoding genes that harbor miRNA genes within their intronic regions and the possibility that estrogen regulates the expression of transcription factors which control the expression of miRNAs. Collectively, these mechanisms would all lead to an increase in miRNA expression through an increase in the levels/expression of pri-miRNAs. The findings from the current study add an additional mechanism by which steroids may increase the levels of mature miR-NAs. We demonstrate that concurrent exposure to estrogen and progesterone increase expression of Drosha, Dgcr8, Exportin-5 , and Dicer1 with the later two being the most significantly regulated components of the miRNA biogenesis pathway. Induction of Exportin-5 may provide a mechanism by which steroids can increase the shuffling or export of pre-miRNAs to the cytoplasm. Once in the cytoplasm, the exported pre-miRNA population can be processed to the mature miRNA through the increased levels of Dicer1 and then in turn modulate target mRNA stability. In summary, the current study demonstrates for the first time that the expression of the miRNA biogenesis components Drosha, Dgcr8, Exportin-5 , and Dicer1 is significantly up-regulated by ovarian steroids in mouse uterine tissue and that this modulation occurs through their respective cognate receptor pathways. Steroidal modulation of these enzymes may provide an additional level of regulation of miRNA generation which in turn could impact uterine gene expression and uterine physiology.

All animal studies were approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee (IACUC). Mature female mice (CD1; Harlan Laboratories, Inc., Indianapolis, IN, USA), 2–4 months of age, were ovariectomized, rested 2 weeks and treated s.c. with either vehicle (0.1 ml sesame oil), estradiol-17 β (E 2 , 10 μg/kg BW), progesterone (P 4 , 100 mg/kg BW) or E 2 + P 4 (previous doses). Mice were then killed at 0 h (no treatment), 4, 8, and 24 h after injection. Mice were killed by cervical dislocation and uteri were removed and trimmed of fat. One-half of each uteri was placed into RNalater (Ambion, Austin, TX, USA) at −20 °C until processed for RNA isolation while the remaining half was snap-frozen in liquid nitrogen until utilized for protein extraction. A second study was conducted to examine whether steroidal regulation of uterine miRNA biogenesis components was mediated through the estrogen and progesterone nuclear receptors. To do so, mice were injected s.c. with estrogen receptor antagonist ICI-182,780 (ICI; Tocris Cookson Inc., Ellisville, MO; 20 mg/kg dissolved in 100% ethanol and resuspended in sesame oil) or the progesterone receptor antagonist RU486 (Mifepristone; Dr. A. F. Parlow, NIDDK’s National Hormone and Pituitary Program, Torrance, CA; 20 mg/kg dissolved in 100% ethanol and resuspended in sesame oil) 30 min before each steroidal treatment. Additional groups consisted of mice receiving only vehicle, steroid alone (previous doses), or receptor antagonist alone. All mice were killed 8 h after steroid or vehicle administration. We selected the 8-h time point as this had been shown in the first study to be the optimal time point for both E 2 and P 4 induction of miRNA biogenesis components as well as a time point for significant increase in miR-451 expression. Quantitative RT-PCR (qRT-PCR) was performed as previously described [ 39 ]. Briefly, total RNA was isolated using miRNA easy kits (Qiagen, Valencia, CA, USA) according to recommendations of the manufacturer. Total RNA (250 ng in 5 μl) was reverse transcribed using reverse transcription (RT) kits (Applied Biosystems; Foster City, CA, USA) following the manufacturer’s protocol with the following modifications. Briefly, mRNAs were reverse transcribed in a single reaction using 2 μl of each mRNA specific 5 × RT primers. Primers for the miRNA biogenesis components Drosha, DGCR8, Exportin-5 , and Dicer1 were designed using Primer Express 3.0 software and synthesized by Integrated DNA Technology (IDT, Coralville, IA, USA; see Table 1 ). Resulting material was then used for independent qRT-PCR for each mRNA. RT-PCR was carried out on an Applied Biosystems HT7900 Sequence Detector. In order to account for differences in starting material, human 18S primers and probe reagents were used for miRNA biogenesis components while U6 was used for miR-451 . Primers for both U6 and miR-451 were purchased from Applied Biosystems. A standard curve was run in each assay, with an arbitrary value assigned to the highest standard and corresponding values to the subsequent dilutions. Each cDNA sample was run in triplicate and the relative abundance of each target divided by the relative abundance of 18S (or U6) to normalize for the starting quantity of cDNA. Each primer set included a minus RT control. The delta–delta CT method was used to calculate the fold-change values among samples. Western analysis was performed as previously described [ 39 ]. Briefly, total protein was extracted from frozen uteri using RIPA buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS) containing a protease/phosphatase inhibitor cocktail (0.1 mg/ml PMSF, 30 μl/ml aprotinin, 5 μg/ml leupeptin, 1 mM sodium orthovanadate; Sigma, St. Louis, MO, USA). Protein concentration in each sample was determined using the DC Protein Assay (Bio-Rad Laboratories, Richmond, CA, USA). The same amount of protein (40 μg) was subjected to 4–12% Bis–Tris (Invitrogen, Carlsbad, CA, USA) gel electrophoresis and electroblotted onto nitrocellulose membranes (Bio-Rad Laboratories, Richmond, CA, USA). Rabbit anti-Dicer1 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-Exportin-5 (1:500; Santa Cruz Biotechnology, Inc.), and goat anti-rabbit secondary antibody (1:5000, Jackson Immunoresearch Laboratories, Inc., Westgrove, PA, USA) were used. Stripping and reprobing for β -actin (Santa Cruz Biotechnology, Inc.) was conducted to normalize protein expression levels. Immunodetection was carried out using an enhanced chemiluminescence (ECL) kit (Amersham Biosciences, Piscataway, NJ, USA). Expression of each protein was normalized to that of β -actin and expressed as a fold-change from vehicle. Unless otherwise indicated, all data were analyzed by one-way ANOVA for comparison across time points. When an F test indicated statistical significance, post-hoc analysis was made using the Tukey HSD procedure. Significance was set at P < 0.05 for all comparisons.