Expression data from primary culture human myometrial cells
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ABSTRACT: Inflammation plays a central role in many human diseases. Human parturition also resembles an inflammatory reaction, where progesterone (P4) and progesterone receptors (PRs) have already been demonstrated to suppress contraction-associated gene expression. In our previous studies, we have found that the progesterone actions, including progesterone-induced gene expression and progesterone’s anti-inflammatory effect, are mediated by PR, GR or both. In this study, we used microarrays to find P4 and IL-1β responsive genes and which IL-1β responsive genes were repressed by P4. These data may provide a broader view of gene networks and cellular functions regulated by P4 and IL-1βin human myometrial cells. In our future study, this will also help us understand the role of PR and GR in human parturition. Primary cultures of human myometrial cells were grown from myometrial biopsies obtained at the time of elective caesarean section. Cells were exposed to different stimuli, IL-1β (5ng/mL) and P4 (10 µM), either alone or in combination for 6 h, and then total RNA were extracted from each culture. Three comparisons were carried out including: 1. V vs. P4; 2. V vs. IL-1β; 3. IL-1β vs. IL-1β+P4.
Project description:Inflammation plays a central role in many human diseases. Human parturition also resembles an inflammatory reaction, where progesterone (P4) and progesterone receptors (PRs) have already been demonstrated to suppress contraction-associated gene expression. In our previous studies, we have found that the progesterone actions, including progesterone-induced gene expression and progesterone’s anti-inflammatory effect, are mediated by PR, GR or both. In this study, we used microarrays to find P4 and IL-1β responsive genes and which IL-1β responsive genes were repressed by P4. These data may provide a broader view of gene networks and cellular functions regulated by P4 and IL-1βin human myometrial cells. In our future study, this will also help us understand the role of PR and GR in human parturition.
Project description:During pregnancy, the myometrium remains quiescent but at term, switches to a state capable of producing a series of coordinated contractions for the delivery of the fetus. Myometrial contractions of labour signify the normal physiological end-point of pregnancy but the biochemical onset of labour may occur at or before term via a series of changes in expression of labour associated genes that are responsible for controlling the activity of the uterus during pregnancy and parturition. There is increasing evidence that components of the cAMP-signalling pathway are up-regulated in the human myometrium during pregnancy to promote the relaxation of the myometrium until term. Our aim was to determine which cAMP-associated genes are important during pregnancy and parturition by exposing myometrial cells to forskolin and performing an a gene array. We then plan to study the trend of the cAMP-associated genes at different stages of gestation and during labour. In this study, we used microarrays to elucidate forskolin responsive genes in human myometrium. These data may provide a broader view of gene networks and cellular functions regulated by forskolin in human myometrial cells. In our future study, this will also help us understand the role of cAMP in human parturition. Primary cultures of human myometrial cells were grown from myometrial biopsies obtained at the time of elective caesarean section at term. Cells were exposed to forskolin (100 µM) for 48 hours, and then total RNA were extracted from each culture. Two comparisons were carried out including: 1. Control 2. Forksolin
Project description:Circulating progesterone (P4) levels decline before the onset of parturition in most animals, but not in humans. This has led to the suggestion that there is functional withdrawal of P4 action at the myometrial level prior to labor onset. Mifepristone is widely used to induce human labour In this study, we aimed to establish and validate a model of human myometrial explants for the study of P4 action. Myometrial biopsies obtained at Caesearean section at term were dissected into explants after a portion was immediately snap-frozen (t=0). Transcriptomic comparison of paired explants and primary myometrial cells as well as the hTert immortalized myometrial cell line demonstrated that explants more closely resemble t=0. Biopsies obtained from non-laboring women at elective Caesarean section at term were divided into 3: (i) dissected and immediately snap-frozen (t=0), (ii) dissected into 3x3x3mm3myometrial explants and (iii) processed for primary cell culture. Explants, primary cells at passage 4 (the typical passage our group uses for experiments) and hTERT cells were cultured for a period of 30 hours without treatment. Total RNA was extracted and microarray analysis performed. 6 replicates were used for this study.
Project description:Parturition involves cellular signaling changes driven by the complex interplay between progesterone (P4), inflammation, and the cyclic adenosine monophosphate (cAMP) pathway. To characterize this interplay, comprehensive transcriptomic studies utilizing 8 treatment combinations were performed on the hTERT-HMA/B telomerase immortalized human myometrial cell line and myometrium obtained from term c-section deliveries. Genome-wide RNA-sequencing was performed on total RNA extracted from hTERT-HMA/B cells treated with all combinations of P4, forskolin (induces cAMP), and interleukin-1β (IL-1β). Gene set enrichment and regulatory network analyses was then used to identify pathways commonly, differentially, or synergistically regulated by each treatment. Tissue similarity index (TSI) was also applied to characterize the correspondence between cell lines and tissue phenotypes. In addition to their individual anti-inflammatory effects, P4 and cAMP synergistically blocked specific inflammatory pathways/regulators including STAT3/6, CEBPA/B, and OCT1/7, but not NFKB. TSI analysis indicated that forskolin+P4- and IL-1β-treated cells exhibit transcriptional signatures highly similar to non-laboring and laboring term myometrium, respectively. The data identify potential therapeutic targets to prevent preterm birth and show that the hTERT-HMA/B cell line provides is an accurate transcriptional model for term ± labor myometrium.
Project description:Circulating progesterone (P4) levels decline before the onset of parturition in most animals, but not in humans. This has led to the suggestion that there is functional withdrawal of P4 action at the myometrial level prior to labor onset. However, to date, no evidence of a loss of P4 function has been provided. Mifepristone is a mixed progesterone and glucocorticoid receptor antagonist that is frequently used to induce human labor. However, its effect on the myometrial tissue transcriptome hs not been determined. In this study, we aimed to identify the effects of mifepristone treatment on the pregnant myometrium at term in order to better understand the possible mechanisms underlying the loss of P4 function in human labor.
Project description:Circulating progesterone (P4) levels decline before the onset of parturition in most animals, but not in humans. This has led to the suggestion that there is functional withdrawal of P4 action at the myometrial level prior to labor onset. Mifepristone is widely used to induce human labour In this study, we aimed to establish and validate a model of human myometrial explants for the study of P4 action. Myometrial biopsies obtained at Caesearean section at term were dissected into explants after a portion was immediately snap-frozen (t=0). Transcriptomic comparison of paired explants and primary myometrial cells as well as the hTert immortalized myometrial cell line demonstrated that explants more closely resemble t=0.
Project description:Timely control of parturition is crucial for maternal and fetal health. Failures on this biological process often result in pregnancy complications including preterm birth, labor dystocia, and health disorders on newborn babies. The myometrium is the muscular structure of the uterus maintaining uterine structural integrity and providing contractile force for parturition. The myometrial structure changes in adaptation to the pregnancy via stage-specific transcriptomic profiles. Progesterone signaling plays a crucial role in myometrial remodeling. The present study profiles the transcriptome of human myometrial specimens that are expected to manifest a wide spectrum of progesterone signaling via the RNAseq assay.
Project description:Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was utilized to analyze adenomyotic and normal myometrial tissues from ten AM patients who underwent hysterectomy with myometrial fibrosis confirmed by Masson staining. This analysis established comprehensive proteomic profiles of adenomyosis patients and revealed widespread alterations in the proteome and metabolome within normal and fibrotic myometrium. Key proteins and signaling pathways linked to myometrial fibrogenesis were identified based on proteomic data.
Project description:The myometrium is an important reproductive tissue composed primarily of smooth muscle cells. Contractility of the myometrial smooth muscle cells during pregnancy and labour is modulated by hormones. Despite much research, little is known about the molecular mechanism by which estrogen and progesterone regulate myometrial contractility. This study investigates global gene expression profile of cultured human uterine smooth muscle cells (hUtSMCs) following 17β-estradiol (E2) and/or progesterone treatments using cDNA microarray technology. Furthermore the effects of addition of the progesterone inhibitor RU486 were investigated. Many genes were regulated in the presence of P4 or E2 alone but almost six times these numbers were regulated in the presence of the combination. The majority of these genes returned to near baseline levels (control samples) on addition of RU486. In total 796 annotated genes were significantly differentially expressed in the combined presence of P4 and E2 (relative to untreated levels). Furthermore, 666 annotated genes were significantly regulated by the addition of RU486 to the P4/E2 combination (relative to P4/E2 levels). Gene ontology analysis of these differentially expressed genes revealed a strong association of P4/E2 treatment with the downregulated expression of genes involved in cell communication, signal transduction, channel activity, inflammatory response and differentiation. Upregulated processes included cell survival (anti-apoptosis), gene transcription, steroid hormone biosynthesis, muscle development, insulin receptor signaling and cell growth. Functional withdrawal of progesterone using RU486 effectively reversed the processes induced by P4/E2 treatment. The hUtSMC system is an additional useful model to investigate steroid effects on smooth muscle cells in isolation from other myometrial cell types. In this study human myometrial smooth muscle cells (hUtSMCs) purchased from Lonza were cultured and treated with 17beta estradiol and progesterone either alone or in combination. In addition the 17beta estradiol and progesterone combined treated samples were also treated with the progesterone inhibitor RU486 (mifepristone). RNA was isolated after 72h from both treated and untreated control cells. The experiment was repeated 3 times. Please note that slides were scanned three times successively (i.e. 3 .gpr files per sample) and the median raw values were used as input raw data for GeneSpring analysis (Median_Table_InputRAW.xlsx).
Project description:In chorioamnionitis (CAM), a major cause of preterm birth (PTB), maternal-fetal inflammatory responses in the decidua and amnio-chorion cause the release of cytokines that elicit cervical ripening, fetal membrane rupture and myometrial activation. We posited that this inflammatory milieu can trigger PTB via inhibited progesterone receptor (PR) expression and increased decidual prostaglandin (PG) production. We found significantly lower decidual cell PR levels in CAM-complicated PTB using immunohistochemistry. Decidual cells (DCs) treated with IL-1β displayed decreased PR expression and significantly increased PGE2 and PGF2α production and COX2 expression. While addition of PGF2α to DC cultures was also found to suppress PR expression, the COX inhibitor, indomethacin, did not reverse IL-1β suppression of PR expression in DC cultures. Although IL-1β treatment activated NF-B, ERK1/2 and p38 MAPK signaling cascades in DCs, only inhibition of ERK1/2 MAPK signaling completely reversed IL-1β suppressed PR levels. These findings suggest that CAM-associated PTB is induced at least in part by IL-1β-mediated functional progesterone withdrawal.