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Profile of MicroRNA Expression in Endometrial Cell during In Vitro Culture According to Progesterone Concentration.

ABSTRACT: Artificial uterus using endometrium implant can be a novel treatment strategy for infertile women with refractory endometrial dysfunction. At early pregnancy, the function of uterine endometrial cells for the communication between the conceptus of pre-implantation period and maternal reproductive system is essential. MicroRNA (miR) expression profile of endometrial cells according to progesterone, a crucial pregnancy-maintaining hormone, provides important data for in vitro endometrial cell culture strategy that is useful for engineering artificial uteri using endometrial implants. The present study aimed to evaluate the miR expression profile of in vitro cultured endometrial cells under hormonal milieu mimicking early pregnancy period in terms of progesterone concentration. We cultured murine uterine endometrial cells, human uterine endometrial carcinoma cells, and immortalized human uterine endometrial cells using different progesterone concentrations, and analyzed the expression of miRs critical for early pregnancy. The expression of miR-20a, -21, -196a, -199a, and -200a was differently regulated according to progesterone concentration in different endometrial cell lines. The analysis of candidate target genes showed that the expression of phosphatase and tensin homolog, mucin 1 (MUC1), progesterone receptor, transforming growth factor ? receptor II, matrix metallopeptidase-9 was up-regulated by progesterone treatment in mouse and human endometrial cell lines. These results indicate that physiological concentration range (10-7 and 10-9 M) of progesterone affect the survival and target gene expression via modulating miR expression. Taken together, progesterone can be a crucial factor in regulating miR expression on in vitro cultured endometrial cells.

SUBMITTER: Kim YJ

PROVIDER: S-EPMC6171628 | biostudies-other | 2017 Oct

REPOSITORIES: biostudies-other

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Profile of MicroRNA Expression in Endometrial Cell during <i>In Vitro</i> Culture According to Progesterone Concentration.

Kim Yong Jin YJ Kim Yoon Young YY Kim Dong Won DW Joo Jong Kil JK Kim Hoon H Ku Seung-Yup SY

Tissue engineering and regenerative medicine 20170908 5

Artificial uterus using endometrium implant can be a novel treatment strategy for infertile women with refractory endometrial dysfunction. At early pregnancy, the function of uterine endometrial cells for the communication between the conceptus of pre-implantation period and maternal reproductive system is essential. MicroRNA (miR) expression profile of endometrial cells according to progesterone, a crucial pregnancy-maintaining hormone, provides important data for <i>in vitro</i> endometrial ce ...[more]

PMID: 30603515

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Project description:Study questionWhat doses of secretory phase progesterone (P) in women are associated with altered endometrial structure and/or function?Summary answerConsistently delayed histological maturation was seen at the lowest tested daily P dose (2.5 mg), whereas consistently altered functional response, as reflected by microarray analysis of gene expression was seen at both the 5 and 2.5 mg doses.What is known alreadyProgesterone is absolutely required for normal embryo implantation and pregnancy survival. Progesterone supplementation is beneficial in ART cycles.Study design, size, durationIn this case-control experimental trial, 46 healthy young female volunteers (age 19-34) underwent a single modeled endometrial cycle after GnRH down-regulation or monitored in natural cycles.Participants/materials, setting, methodsIn a university hospital, modeled cycles were obtained by GnRH agonist down-regulation, transdermal estradiol (E2) (0.2 mg/d), and daily injections of P in oil for 10 days: 2.5 mg (n = 6), 5 mg (n = 6), 10 mg (n = 12) or 40 mg (n = 12), after the 10th day of E2. Ten healthy, ovulatory women were used as controls. Endometrial biopsies were obtained on the 10th day of P exposure, or urinary LH surge (in controls). Analysis included histological dating, serum progesterone levels, microarray analysis of the whole genome, RT-PCR, western blot and comparison with the GEO database.Main results and the role of chanceIn endometrial biopsies, a morphological delay appears in the 2.5 mg/day of P group. Higher sub-physiological levels of P (≥5 mg/day) resulted in normal histology, but aberrant gene expression. P levels required for consistent histological delay were lower than those in all ovulatory women. Gene expression abnormalities occurred at higher sub-physiological P concentrations, without a change in histology, a functional-morphological disassociation. The expression of some endometrial receptivity-associated genes appeared multiphasic, with peak or nadir of mean or median expression levels between the lowest and highest doses, suggesting sustained supraphysiological doses seen in ART treatment cycles may not be optimal.Large scale dataGEO DataSets ID: 200056980; GSE 56980.Limitations, reasons for cautionThese results were obtained in fertile women, who may respond differently from infertile subjects.Wider implications of the findingsThe dose of P required for normal endometrial structure (5 mg/day) corresponds to a P concentration well below that seen in ovulatory women, suggesting that persistently delayed mid-secretory histology cannot be solely due to inadequate P concentrations in an ovulatory cycle. Endometrial gene expression is differentially regulated by different doses of progesterone. The apparent multiphasic response of some genes to P dose suggests the possibility that P concentration kinetics may play a role in normal endometrial preparation for receptivity. These findings strongly confirm that histologic development is not a reliable measure of endometrial P action.Study funding/competing interest(s)Supported by The Eunice Kennedy Shriver National Institute for Child Health and Disease, National Institute of Health, USA (NICHD/NIH) (R01HD067721 and U54HD30476; SLY and BAL) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 240239/2012-1 (RFS). All authors have no competing interests.

Project description:The expression profile of microRNA (miRNA) in uterine leiomyoma (UL) cells is different from that in normal uterine myometrial (UM) cells. The effect of UL cells on uterine receptivity might vary according to their ability to distort the uterine endometrial cavity. However, the variation in miRNA expression profiles between endometrial cavity-distorting leiomyoma (ECDL) and endometrial cavity non-distorting leiomyoma (ECNDL) cells remains unknown. This study aimed to elucidate whether the expression profile of miRNAs in ECDL cells is dissimilar to that of ECNDL cells in uterus. Pelviscopic myomectomy was performed to obtain tissue samples of UL and their corresponding normal UM tissues (matched) from patients with UL (n = 26), among whom women with ECNDL and ECDL numbered 15 and 11, respectively. The relative expression of hsa-miR-15b, -29a, -29b, -29c, -197, and -200c as well as the candidate target genes in UL cells was compared to those in the matched UM cells using qRT-PCR to assess their ability to cause ECD. The spatial expression of miRNAs and target genes in the UL tissues was analyzed using in situ hybridization. Target gene expression was analyzed using qPCR after transfection with the mimics and inhibitors of miRNAs in UL cells. The relative expression level of miR-15b was upregulated, and the relative expression levels of miR-29a, -29b, -29c, -197, and -200c were downregulated in UL cells compared to those in UM cells. The relative expression levels of progesterone receptor, estrogen receptor, and matrix metalloproteinases (MMPs) were upregulated in UL cells compared to those in UM cells. The relative expression levels of miR-29c and -200c were downregulated, and the relative expression levels of estrogen receptor, MMPs and tissue inhibitors of metalloproteinases (TIMPs) were upregulated in ECDL cells compared to those in ECNDL cells. The expression profile of miRNAs in UL cells varied with respect to the occurrence or absence of endometrial cavity distortion. The biochemical properties of UL might be regulated by miRNAs in order to alter their effect on structural homeostasis of the uterus.

Project description:To design novel therapeutics against bone loss, understanding the molecular mechanisms regulating osteoclastogenesis is critical. Osteoclast formation and function are tightly regulated by transcriptional, post-transcriptional and post-translational mechanisms. This stringent regulation is crucial to prevent excessive or insufficient bone resorption and to maintain bone homeostasis. microRNAs (miRNAs) are key post-transcriptional regulators that repress expression of target mRNAs controlling osteoclast proliferation, differentiation, and apoptosis. Disruption of miRNA-mediated regulation alters osteoclast formation and bone resorption. Prior studies profiled miRNA expression in murine osteoclast precursors treated with RANKL for 24 hours. However, a more complete miRNA signature, encompassing early, mid and late stages of osteoclastogenesis, is wanting. An Agilent microarray platform was used to analyze expression of mature miRNAs in an enriched population of murine bone marrow osteoclast precursors (depleted of B220+ and CD3+ cells) undergoing 1, 3, or 5 days of RANKL-driven differentiation. Expression of 93 miRNAs, changed by >2 fold during early, mid, and late stages of osteoclastogenesis, were identified and sorted into 7 clusters. We validated the function and expression of miR-365, miR-451, and miR-99b, which were found in distinct clusters. Inhibition of miR-365 increased osteoclast number but decreased osteoclast size, while miR-99b inhibition decreased both osteoclast number and size. In contrast, overexpression of miR-451 had no effect. Computational analyses predicted mTOR, PI3 kinase/AKT, cell-matrix interactions, actin cytoskeleton organization, focal adhesion, and axon guidance pathways to be top targets of several miRNA clusters. This suggests that many miRNA clusters differentially expressed during osteoclastogenesis converge on some key functional pathways. Overall, our study is unique in that we identified miRNAs differentially expressed during early, mid, and late osteoclastogenesis in a population of primary mouse bone marrow cells enriched for osteoclast progenitors. This novel data set contributes to our understanding of the molecular mechanisms regulating the complex process of osteoclast differentiation.

Dataset Information

Profile of MicroRNA Expression in Endometrial Cell during In Vitro Culture According to Progesterone Concentration.

Publications

Profile of MicroRNA Expression in Endometrial Cell during <i>In Vitro</i> Culture According to Progesterone Concentration.

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