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MiRNA signature and Dicer requirement during human endometrial stromal decidualization in vitro.

ABSTRACT: Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs) decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human endometrial decidualization.

SUBMITTER: Estella C

PROVIDER: S-EPMC3401238 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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miRNA signature and Dicer requirement during human endometrial stromal decidualization in vitro.

Estella Carlos C Herrer Isabel I Moreno-Moya Juan Manuel JM Quiñonero Alicia A Martínez Sebastián S Pellicer Antonio A Simón Carlos C

PloS one 20120720 7

Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human diso ...[more]

PMID: 22911744

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Project description:Nucleotide-binding oligomerization domain (NACHT), leucine rich repeat (LRR) and pyrin domain (PYD) 7 containing protein, NLRP7, is a member of the NLR family which serves as innate immune sensors. Mutations and genetic variants of NLRP7 have been found in women with infertility associated conditions, such as recurrent hydatidiform mole, recurrent miscarriage, and preeclampsia. Decidualization of endometrial stromal cells is a hallmark of tissue remodeling to support embryo implantation and proper placental development. Given defective decidualization has been implicated in miscarriage as well as preeclampsia, we aimed to explore the link between the NLRP7 gene and decidualization.Endometrial samples obtained from pregnant women in the first trimester and non-pregnant women were used to study NLRP7 expression pattern. The human telomerase reverse transcriptase (hTERT)-immortalized human endometrial stromal cells (T-HESCs) were used to study the effect of NLRP7 on decidualization. Decidualization of T-HESCs was induced with 1 μM medroxyprogesterone acetate (MPA) and 0.5 mM 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP). siRNA was used to knock down NLRP7 while lentiviral vectors were used to overexpress NLRP7 in cells. NLRP7 expression was detected by immunofluorescence, qRT-PCR, and Western blotting. Decidualization markers, Insulin-like growth factor-binding protein 1 (IGFBP-1) and prolactin (PRL), were detected by qRT-PCR and ELISA. Nuclear translocation of NLRP7 was detected by the subcellular fractionation and confocal microscopy. The effect of NLRP7 on progesterone receptor (PR) activity was evaluated by a reporter system.NLRP7 was up-regulated in the decidual stromal cells of human first-trimester endometrium. After in vitro decidualization, T-HESCs presented with the swollen phenotype and increased expressions of IGFBP-1 and PRL. Knockdown or over-expression of NLRP7 reduced or enhanced the decidualization, respectively, according to the expression level of IGFBP-1. NLRP7 was found to translocate in the nucleus of decidualized T-HESCs and able to promote PR activity.NLRP7 was upregulated and translocated to the nucleus of the endometrial stromal cells in an in vitro decidualization model. Overexpressed NLRP7 promoted the IGFBP-1 expression and PR reporter activation. IGFBP-1 expression decreased with the knockdown of NLRP7. Therefore, we suggest that NLRP7 contributes to in vitro decidualization of endometrial stromal cells.

Project description:Multiple studies have shown associations between exposure to endocrine disrupting chemicals (EDCs) and reduced fertility in women. However, little is known about the target organs of chemical disruption of female fertility. Here, we focus on the hormone-sensitive uterine lining, the endometrium, as a potential target. Decidualization is the morphological and functional change that endometrial stromal cells undergo to support endometrial receptivity, which is crucial for successful implantation, placentation, and pregnancy. We investigated the effect of nine selected EDCs on primary human endometrial stromal cell decidualization in vitro. The cells were exposed to a decidualization-inducing mixture in the presence or absence of 1 μM of nine different EDCs for nine days. Extent of decidualization was assessed by measuring the activity of cAMP dependent protein kinase, Rho-associated coiled-coil containing protein kinase, and protein kinase B in lysates using photoluminescent probes, and secretion of prolactin into the media by using ELISA. Decidualization-inducing mixture upregulated activity of protein kinases and prolactin secretion in cells derived from all women. Of the tested chemicals, dichlorodiphenyldichloroethylene (p,p'-DDE), hexachlorobenzene (HCB) and perfluorooctanesulfonic acid (PFOS) significantly reduced decidualization as judged by the kinase markers and prolactin secretion. In addition, bisphenol A (BPA) reduced prolactin secretion but did not significantly affect activity of the kinases. None of the EDCs was cytotoxic, based on the assessment of total protein content or activity of the viability marker casein kinase 2 in lysates. These results indicate that EDCs commonly present in the blood circulation of reproductive-aged women can reduce decidualization of human endometrial stromal cells in vitro. Future studies should focus on detailed hazard assessment to define possible risks of EDC exposure to endometrial dysfunction and implantation failure in women.

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Dataset Information

MiRNA signature and Dicer requirement during human endometrial stromal decidualization in vitro.

Publications

miRNA signature and Dicer requirement during human endometrial stromal decidualization in vitro.

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