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Transcriptome of human decidual cells treated by siRNA targeting FOXO1

ABSTRACT: Here we report the gene expression profile of in vitro cultured human endometrial stromal cells treated with siRNA targeting FOXO1 piror to eutherian differentiation media exposure. The eutherian differentiation media contains cyclic AMP (cAMP) analogue 8-Br-cAMP and the progesterone (P4) analogue medroxyprogesterone acetate (MPA).

ORGANISM(S): Homo sapiens

PROVIDER: GSE115832 | GEO | 2018/06/21

REPOSITORIES: GEO

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Project description:In the current study, we hypothesized that if bone-marrow derived MSC contribute to endometrial regeneration and are progenitors to hESF, their treatment with agents known to regulate hESF differentiation could promote their differentiation down the stromal fibroblast lineage. To this end, we treated bone marrow-derived MSC with estradiol (E2) and progesterone (P4), BMP2, and activators of the PKA pathway and investigated specific markers of hESF differentiation (decidualization). Furthermore, we investigated the transcriptome of these cells in response to cAMP and compared this to the transcriptome of hESF decidualized in response to activation of the PKA pathway. The data support that MSC can be differentiated down the hESF pathway, as evidenced by changes in cell shape and common expression of decidual markers and other genes important in hESF differentiation and function. Bone marrow-derived MSC were expanded in DMEM (high glucose) medium containing 10% fetal bovine serum, 1% penicillin and streptomycin. At 80% confluency MSC were trypsinized and plated at 5x103 cells/cm2 in 6cm plates. Nearly confluent cells were serumstarved overnight and thereafter treated for 0, 3, 7, 14 and 21 days in low-serum medium (2% FBS) with the following treatments: (a) 10nM E2/1mM P4; (b) 0.5mM or 1mM 8-Br-cAMP (cAMP); (c) 50nM or 100nM BMP-2; (d) 0.5mM and 1mM 8-Br-cAMP + BMP-2; (d) 0.5mM and 1mM 8-BrcAMP + BMP-2 + E2/P4; (e) 50nM or 100nM human FSH, with vehicle controls at each time point. Each experiment was conducted in duplicate and repeated at least 3 times. Media were changed every third day. Endpoints were prolactin (PRL) and IGF binding protein-1 (IGFBP1) mRNA, assessed by quantitative real-time RT-PCR, and protein in conditioned media, assessed by ELISA (see below), after 3, 7, 14 and 21 days of incubation. Immunostaining for vimentin, cytokeratin 7, and Oct-4 was performed before and after MSC treatments to assess the stromal lineage of MSC and their differentiation status. MSC expressed vimentin and did not express cytokeratin or Oct-4, independent of culture conditions or duration of culture or treatment. The morphological appearance of MSC under different treatment conditions was photographed using a Leica CTR 6500 microscope. Microarray analysis was performed with MSC, n=3, at t=0 (untreated, uncultured control), 14day vehicle control, and 14day treated with 8-Br-cAMP. Only high quality samples (RIN=9.1-10) were used for microarray analysis. Hybridization was performed with Affymetrix Human Gene 1.0 ST arrays.

Project description:Use of long-acting progestin-only contraceptives (LAPCs) offers a discrete and highly effective family planning method. Abnormal uterine bleeding (AUB) is the major side effect of, and cause for, discontinuation of LAPCs. The endometria of LAPC-treated women display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow and oxidative stress. To understand the mechanisms underlying AUB, we propose to identify LAPC-modulated unique gene cluster(s) in human endometrial stromal cells (HESCs). Protein and RNA isolated from cultured HESCs treated 7 days with estradiol (E2) or E2+medroxyprogesterone acetate (MPA) or E2+ etonogestrel (ETO) or E2+progesterone (P4) were analyzed by q-PCR and immunoblotting. HSCORES were determined for immunostained-paired endometria of pre-and 3 months post-Depo-Provera (Depo) treated women (n=6) and ovariectomized guinea pigs (GPs; n=12) treated with placebo or E2 or MPA or E2+MPA for 21 days. In HESCs, whole genome analysis identified a 67-gene group regulated by all three progestins, whereas a 235-gene group was regulated by E2+ETO and E2+MPA, but not E2+P4. Ingenuity pathway analysis identified glucocorticoid receptor (GR) activation as an upstream regulator of the 235 LAPCMPA and ETO-specific genes. Among these, microarray results demonstrated significant enhancement of FKBP51, a repressor of PR/GR transcriptional activity, by both LAPCsMPA and ETO. q-PCR and immunoblot analysis confirmed the microarray results. In endometria of post-Depo versus pre-Depo administered women, FKBP51 expression was significantly increased in endometrial stromal and glandular cells. In GPs, E2+MPA or MPA significantly increased FKBP51 immunoreactivity in endometrial stromal and glandular cells versus placebo- and E2-administered groups. LAPC MPA or ETO administration activates GR signaling and increases endometrial FKBP51 expression, which could be one of the mechanisms causing AUB by inhibiting feeds back to inhibit PR- and GR-mediated transcription. The resultant PR- and/or GR-mediated functional withdrawal may contribute to associated endometrial inflammation, aberrant angiogenesis, and bleeding.

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Transcriptome of human decidual cells treated by siRNA targeting FOXO1

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