scholarly journals 254.Interleukin-11 enhances endometrial stromal cell decidualisation via activation and inhibition of target genes

2004 ◽  
Vol 16 (9) ◽  
pp. 254
Author(s):  
C. A. White ◽  
E. Dimitriadis ◽  
A. Sharkey ◽  
C. J. Stoikos ◽  
L. A. Salamonsen
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Stromal Cells ◽  
Target Genes ◽  
Fold Change ◽  
Rt Pcr ◽  
Endometrial Stromal Cells ◽  
Cytoplasmic Staining ◽  
Decidual Cells ◽  
Custom Made

Differentiation of endometrial stromal cells into decidual cells is essential for successful embryo implantation. Interleukin (IL)-11 signalling is required for decidualisation in the mouse (1,2) and the expression pattern of IL-11 and its receptors during the menstrual cycle suggests a role for IL-11 in human decidualisation (3). Exogenous IL-11 has been shown to enhance hormone-induced decidualisation of human endometrial stromal cells in culture (4). This study aimed to determine the effects of IL-11 on downstream gene expression in endometrial stromal cells following 12 days of progesterone-induced decidualisation, and to examine the expression and functional significance of IL-11 target genes during this process. Stromal cells isolated from endometrial biopsies (n = 6) were decidualised with 17β-oestradiol and medroxyprogesterone acetate (EP) or EP with 100 ng/mL recombinant human IL-11. Medium was changed every 48 h, and total RNA extracted on Day 12 for gene expression analysis using custom-made 15K cDNA microarrays. Quantitative real-time RT-PCR was performed on the same samples to confirm gene expression levels. In subsequent experiments (n = 2), cells were cytocentrifuged onto glass slides for immunocytochemistry using specific antibodies. Microarray analysis revealed 16 upregulated and 11 downregulated cDNAs in EP + IL-11 compared to EP treated cells. Among these were IL-1β (6.1-fold upregulated) and insulin-like growth factor binding protein (IGFBP)-5 (3.6-fold downregulated). Using real-time RT-PCR, IL-11 was confirmed to increase IL-1β (fold change 1.3–107.1) and decrease IGFBP-5 (fold change 2.8–469.0) transcript abundance in 6 patients. Immunolocalisation of IL-1β in EP and EP + IL-11 treated cells revealed more intense vesicular cytoplasmic staining with IL-11 treatment, while staining intensity for IGFBP-5 was not affected. Interactions between IL-11 and its downstream targets IL-1β and IGFBP-5 are likely to have functional importance in early pregnancy, and may provide novel targets for the manipulation of human fertility. (1) Robb L, Li R, Hartley L, Nandurkar HH, Koentgen F, Begley CG (1998) Nat. Med. 4, 303–308. (2) Bilinski P, Roopenian D, Gossler A (1998) Gene Dev. 12, 2234–2243. (3) Dimitriadis E, Salamonsen LA, Robb L (2000) Mol. Hum. Reprod. 6, 907–914. (4) Dimitriadis E, Robb L, Salamonsen LA (2002) Mol. Hum. Reprod. 8, 636–643.

410. Gene expression analysis in endometriotic lesions using laser capture microdissection

2008 ◽  
Vol 20 (9) ◽  
pp. 90
Author(s):  
L. Fu ◽  
J. E. Girling ◽  
P. A. W. Rogers
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Laser Capture Microdissection ◽  
Stromal Cells ◽  
Expression Profiles ◽  
Specific Gene ◽  
Rt Pcr ◽  
Normal Endometrium ◽  
Rna Quality ◽  
Laser Capture

Previous studies examining gene expression profiles in normal endometrium and endometriotic lesions have used RNA extracted from whole tissue samples. Results from these studies can be difficult to interpret as they reflect expression averaged across several different cell types that may be functionally quite different. The aim of this study was to establish laser capture microdissection (LCM) as a technique to examine gene expression in stromal and epithelial cells from normal and ectopic endometrium. We hypothesised that genes associated with inflammation would be elevated in cells from endometriotic lesions. Full thickness uterine samples were collected during abdominal hysterectomy from normal cycling premenopausal women. Endometriotic lesions were collected during abdominal laparoscopy. Samples were either frozen in OCT or stored in RNAlater for 12 h before freezing. Tissues were immunostained with an antibody against CD10 to identify ectopic endometrial stromal cells before LCM. Endometrial epithelial and stromal cells were collected using the PALM MicroLaser System. RNA quality was accessed using Experion. TGFβ1, MMP1, αSMA, SMAD2 and NFκB mRNA was analysed using real-time RT–PCR. Of the endometriotic samples stored in OCT (n = 58), only 14% (n = 8) had visible endometrial glands. Of these, only 37% (n = 3) had RNA of an acceptable quality for further analysis. However, RNA quality and quantity were dramatically improved in 3 of 5 samples collected in RNAlater. In preliminary studies, expression of TGFβ1 and αSMA mRNA was elevated in endometriotic lesions in comparison to the normal endometrium, whereas NFκB expression did not change. We have shown that RNAlater solution is useful to preserve RNA quality for small clinical endometriotic samples and that immuno-guided LCM-generated homogenous cell populations coupled with real-time RT–PCR can provide valuable insights into cell and disease-specific gene expression in endometriotic lesions.

Statistical models in assessing fold change of gene expression in real-time RT-PCR experiments

2006 ◽  
Vol 30 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Wenjiang J. Fu ◽  
Jianbo Hu ◽  
Thomas Spencer ◽  
Raymond Carroll ◽  
Guoyao Wu
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Statistical Models ◽  
Fold Change ◽  
Rt Pcr

scholarly journals Hsa-miR-222 Is Involved in Differentiation of Endometrial Stromal Cells in Vitro

Endocrinology ◽  
2009 ◽  
Vol 150 (10) ◽  
pp. 4734-4743 ◽  
Author(s):  
Kun Qian ◽  
Linli Hu ◽  
Hong Chen ◽  
Haixia Li ◽  
Na Liu ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Target Genes ◽  
Embryo Implantation ◽  
Endometrial Stromal Cells ◽  
Protein Levels ◽  
Mirna Genes ◽  
False Discovery ◽  
Decidual Cells ◽  
Expression Variance

Abstract Decidualization is a critical step during embryo implantation and characterized by the differentiation of endometrial stromal cells (ESCs) into decidual cells. Because miRNAs are important determinants of cellular fate specification, in this study, the miRNA expression in ESCs during in vitro decidualization was profiled by using a microarray. Significance analysis of microarrays revealed that 49 miRNA genes were differently (>2-fold) expressed between the noninduced ESCs and induced ESCs with a false discovery rate of 0. The expression variance of hsa-miR-222, 221, 143, 101, 30d, 30c, 181b, 27b, 29b, 507, and 23a was validated by using quantitative PCR (P < 0.05). Based on microRNA (miRNA) and mRNA expression variance and predicted target genes of miRNAs, a bioinformatic model of miRNAs controlling ESCs differentiation was formulated. Finally, we proved that down-regulation of has-miR-222 could decrease the number of cells in S phase during ESCs differentiation (P < 0.05). Antisense oligonucleotides of has-miR-222 could increase reporter gene expression by targeting the 3′ untranslated regions of CDKN1C/p57kip2 mRNAs as well as increase CDKN1C/p57kip2 protein levels (P < 0.05). In conclusion, our results suggest that a subset of miRNAs play a key role in gene reprogramming during ESCs decidualization and that hsa-miR-222 participates in ESC differentiation by regulating ESCs terminally withdrawing from the cell cycle.

scholarly journals Activation of the protein kinase A pathway in human endometrial stromal cells reveals sequential categorical gene regulation

2003 ◽  
Vol 16 (1) ◽  
pp. 47-66 ◽  
Author(s):  
Emily P. Tierney ◽  
Suzana Tulac ◽  
Se-Te Joseph Huang ◽  
Linda C. Giudice
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Protein Kinase ◽  
Stromal Cells ◽  
Cellular Differentiation ◽  
Cyclin B ◽  
Rt Pcr ◽  
Endometrial Stromal Cells ◽  
Pka Pathway ◽  
Kinase A

Decidualization of endometrial stromal cells is a prerequisite for human implantation and occurs in vivo in response to progesterone and involves activation of the protein kinase A (PKA) pathway. The objective of this study was to determine the molecular signatures and patterns of gene expression during stimulation of this pathway with an analog of cAMP. Endometrial stromal cells from two subjects were treated with or without 8-Br-cAMP (1 mM) for 0, 2, 12, 24, 36, and 48 h and were processed for microarray analysis, screening for 12,686 genes and ESTs. Most abundantly upregulated genes included neuropeptides, immune genes, IGF family members, cell cycle regulators, extracellular matrix proteases, cholesterol trafficking, cell growth and differentiation, hormone signaling, and signal transduction. Most abundantly downregulated genes included activator of NF-κB, actin/tropomyosin/calmodulin binding protein, cyclin B, IGFBP-5, α1 type XVI collagen, lipocortin III, l-kynurenine hydrolase, frizzle-related protein, and cyclin E2. RT-PCR validated upregulation of IGFBP-1, preprosomatostatin, and IL-11, and Northern analysis validated their kinetic upregulation. RT-PCR confirmed downregulation of IGFBP-5, cyclin B, and TIL-4. K-means analysis revealed four major patterns of up- and downregulated genes, and genes within each ontological group were categorized into these four kinetic patterns. Within each ontological group different patterns of temporal gene expression were observed, indicating that even genes within one functional category are regulated differently during activation of the PKA pathway in human endometrial stromal cells. Overall, the data demonstrate kinetic reprogramming of genes within specific functional groups and changes in genes associated with nucleic acid binding, cell proliferation, decreased G protein signaling, increased STAT pathway signaling, structural proteins, cellular differentiation, and secretory processes. These changes are consistent with cAMP modulating early events (0–6 h) primarily involving cell cycle regulation, subsequent events (12–24 h) involving cellular differentiation (including changes in morphology and secretory phenotype), and late events (24–48 h) mediating more specialized function, including immune modulators, in the human endometrial stromal cell.

scholarly journals Optimizations for identifying reference genes in bone and cartilage bioengineering

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Fei Xiong ◽  
Xiangyun Cheng ◽  
Chao Zhang ◽  
Roland Manfred Klar ◽  
Tao He
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Real Time ◽  
Reference Gene ◽  
Muscle Tissue ◽  
Reference Genes ◽  
Target Genes ◽  
The Stability ◽  
And Cartilage

Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) remains one of the best-established techniques to assess gene expression patterns. However, appropriate reference gene(s) selection remains a critical and challenging subject in which inappropriate reference gene selction can distort results leading to false interpretations. To date, mixed opinions still exist in how to choose the most optimal reference gene sets in accodrance to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline. Therefore, the purpose of this study was to investigate which schemes were the most feasible for the identification of reference genes in a bone and cartilage bioengineering experimental setting. In this study, rat bone mesenchymal stem cells (rBMSCs), skeletal muscle tissue and adipose tissue were utilized, undergoing either chondrogenic or osteogenic induction, to investigate the optimal reference gene set identification scheme that would subsequently ensure stable and accurate interpretation of gene expression in bone and cartilage bioengineering. Results The stability and pairwise variance of eight candidate reference genes were analyzed using geNorm. The V0.15- vs. Vmin-based normalization scheme in rBMSCs had no significant effect on the eventual normalization of target genes. In terms of the muscle tissue, the results of the correlation of NF values between the V0.15 and Vmin schemes and the variance of target genes expression levels generated by these two schemes showed that different schemes do indeed have a significant effect on the eventual normalization of target genes. Three selection schemes were adopted in terms of the adipose tissue, including the three optimal reference genes (Opt3), V0.20 and Vmin schemes, and the analysis of NF values with eventual normalization of target genes showed that the different selection schemes also have a significant effect on the eventual normalization of target genes. Conclusions Based on these results, the proposed cut-off value of Vn/n + 1 under 0.15, according to the geNorm algorithm, should be considered with caution. For cell only experiments, at least rBMSCs, a Vn/n + 1 under 0.15 is sufficient in RT-qPCR studies. However, when using certain tissue types such as skeletal muscle and adipose tissue the minimum Vn/n + 1 should be used instead as this provides a far superior mode of generating accurate gene expression results. We thus recommended that when the stability and variation of a candidate reference genes in a specific study is unclear the minimum Vn/n + 1 should always be used as this ensures the best and most accurate gene expression value is achieved during RT-qPCR assays.

scholarly journals Reproductive failure in mice lacking inter-alpha-trypsin inhibitor (ITI) – ITI target genes in mouse ovary identified by microarray analysis

2004 ◽  
Vol 183 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Mika Suzuki ◽  
Hiroshi Kobayashi ◽  
Yoshiko Tanaka ◽  
Naohiro Kanayama ◽  
Toshihiko Terao
Keyword(s):  
Real Time ◽  
Trypsin Inhibitor ◽  
Target Genes ◽  
Reproductive Function ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Reproductive Process ◽  
Female Mice ◽  
Retinoid Metabolism ◽  
Regulatory Effects

Bikunin, a Kunitz-type protease inhibitor, is found in blood and urine. It has been established by two laboratories independently that the bikunin knockout female mice display a severe reduction in fertility: the cumulus oophorus has a defect in forming the extracellular hyaluronan-rich matrix during expansion. Proteins of the inter-alpha-trypsin inhibitor (ITI) family are eliminated in mice in which the bikunin gene has been inactivated, since bikunin is essential for their biosynthesis. Proteins of the ITI family may contribute to the microenvironment in which ovulation takes place. It is not clear, however, whether a single mechanism affects the reproductive function including ovulation. For identifying the full repertoire of the ITI deficiency-related genes, a cDNA microarray hybridization screening was conducted using mRNA from ovaries of wild-type or bik−/− female mice. A number of genes were identified and their regulation was confirmed by real-time RT-PCR analysis. Our screen identified that 29 (0.7%) and 5 genes (0.1%) of the genes assayed were, respectively, up- and down-regulated twofold or more. The identified genes can be classified into distinct subsets. These include stress-related, apoptosis-related, proteases, signaling molecules, aging-related, cytokines, hyaluronan metabolism and signaling, reactive oxygen species-related, and retinoid metabolism, which have previously been implicated in enhancing follicle development and/or ovulation. Real-time RT-PCR analysis confirmed that these genes were up- and down-regulated two- to tenfold by bikunin knockout. These studies demonstrate that proteins of the ITI family may exert potent regulatory effects on a major physiological reproductive process, ovulation.

scholarly journals Progesterone-Dependent Induction of Phospholipase C-Related Catalytically Inactive Protein 1 (PRIP-1) in Decidualizing Human Endometrial Stromal Cells

Endocrinology ◽  
2016 ◽  
Vol 157 (7) ◽  
pp. 2883-2893 ◽  
Author(s):  
Joanne Muter ◽  
Paul J. Brighton ◽  
Emma S. Lucas ◽  
Lauren Lacey ◽  
Anatoly Shmygol ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Stromal Cells ◽  
Nuclear Translocation ◽  
Scaffold Protein ◽  
Trophoblast Invasion ◽  
Differentiation Markers ◽  
Endometrial Stromal Cells ◽  
Decidual Cells ◽  
Phosphoinositide 3 Kinase ◽  
Inactive Protein

Decidualization denotes the transformation of endometrial stromal cells into specialized decidual cells. In pregnancy, decidual cells form a protective matrix around the implanting embryo, enabling coordinated trophoblast invasion and formation of a functional placenta. Continuous progesterone (P4) signaling renders decidual cells resistant to various environmental stressors, whereas withdrawal inevitably triggers tissue breakdown and menstruation or miscarriage. Here, we show that PLCL1, coding phospholipase C (PLC)-related catalytically inactive protein 1 (PRIP-1), is highly induced in response to P4 signaling in decidualizing human endometrial stromal cells (HESCs). Knockdown experiments in undifferentiated HESCs revealed that PRIP-1 maintains basal phosphoinositide 3-kinase/Protein kinase B activity, which in turn prevents illicit nuclear translocation of the transcription factor forkhead box protein O1 and induction of the apoptotic activator BIM. By contrast, loss of this scaffold protein did not compromise survival of decidual cells. PRIP-1 knockdown did also not interfere with the responsiveness of HESCs to deciduogenic cues, although the overall expression of differentiation markers, such as PRL, IGFBP1, and WNT4, was blunted. Finally, we show that PRIP-1 in decidual cells uncouples PLC activation from intracellular Ca2+ release by attenuating inositol 1,4,5-trisphosphate signaling. In summary, PRIP-1 is a multifaceted P4-inducible scaffold protein that gates the activity of major signal transduction pathways in the endometrium. It prevents apoptosis of proliferating stromal cells and contributes to the relative autonomy of decidual cells by silencing PLC signaling downstream of Gq protein-coupled receptors.

scholarly journals The Role of the FOXO1/β2-AR/p-NF-κB p65 Pathway in the Development of Endometrial Stromal Cells in Pregnant Mice under Restraint Stress

2021 ◽  
Vol 22 (3) ◽  
pp. 1478
Author(s):  
Jiayin Lu ◽  
Yaoxing Chen ◽  
Zixu Wang ◽  
Jing Cao ◽  
Yulan Dong
Keyword(s):  
Oxidative Stress ◽  
Stromal Cells ◽  
Restraint Stress ◽  
Target Genes ◽  
Mrna Levels ◽  
Endometrial Stromal Cells ◽  
Protein Levels ◽  
Pregnant Mice

Restraint stress causes various maternal diseases during pregnancy. β2-Adrenergic receptor (β2-AR) and Forkhead transcription factor class O 1 (FOXO1) are critical factors not only in stress, but also in reproduction. However, the role of FOXO1 in restraint stress, causing changes in the β2-AR pathway in pregnant mice, has been unclear. The aim of this research was to investigate the β2-AR pathway of restraint stress and its impact on the oxidative stress of the maternal uterus. In the study, maternal mice were treated with restraint stress by being restrained in a transparent and ventilated device before sacrifice on Pregnancy Day 5 (P5), Pregnancy Day 10 (P10), Pregnancy Day 15 (P15), and Pregnancy Day 20 (P20) as well as on Non-Pregnancy Day 5 (NP5). Restraint stress augmented blood corticosterone (CORT), norepinephrine (NE), and blood glucose levels, while oestradiol (E2) levels decreased. Moreover, restraint stress increased the mRNA levels of the FOXO family, β2-AR, and even the protein levels of FOXO1 and β2-AR in the uterus and ovaries. Furthermore, restraint stress increased uterine oxidative stress level. In vitro, the protein levels of FOXO1 were also obviously increased when β2-AR was activated in endometrial stromal cells (ESCs). In addition, phosphorylated-nuclear factor kappa-B p65 (p-NF-κB p65) and its target genes decreased significantly when FOXO1 was inhibited. Overall, it can be said that the β2-AR/FOXO1/p-NF-κB p65 pathway was activated when pregnant mice were under restraint stress. This study provides a scientific basis for the origin of psychological stress in pregnant women.

scholarly journals si-SNHG5-FOXF2 inhibits TGF-β1-induced fibrosis in human primary endometrial stromal cells by the Wnt/β-catenin signalling pathway

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Limin Liu ◽  
Guobin Chen ◽  
Taoliang Chen ◽  
Wenjuan Shi ◽  
Haiyan Hu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Stromal Cells ◽  
Target Genes ◽  
Cell Model ◽  
Ex Vivo ◽  
Signalling Pathway ◽  
Endometrial Stromal Cells ◽  
Downstream Target ◽  
Related Proteins ◽  
Tgf Β1

Abstract Background Intrauterine adhesions (IUAs) are manifestations of endometrial fibrosis characterized by inflammation and fibrinogen aggregation in the extracellular matrix (ECM). The available therapeutic interventions for IUA are insufficiently effective in the clinical setting for postoperative adhesion recurrence and infertility problems. In this study, we investigated whether si-SNHG5-FOXF2 can serve as a molecular mechanism for the inhibition of IUA fibrosis ex vivo. Methods FOXF2, TGF-β1 and collagen expression levels were measured by microarray sequencing analysis in three normal endometrium groups and six IUA patients. We induced primary human endometrial stromal cells (HESCs) into myofibroblasts (MFs) to develop an IUA cell model with various concentrations of TGF-β1 at various times. Downstream target genes of FOXF2 were screened by chromatin immunoprecipitation combined with whole-genome high-throughput sequencing (ChIP-seq). We investigated ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related proteins in primary HESCs with FOXF2 downregulation by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting (WB), immunohistochemistry (IHC), flow cytometry, ethylenediurea (EdU) and CCK8 assays. We identified long noncoding RNAs (lncRNA) SNHG5 as the upstream regulatory gene of FOXF2 through RNA immunoprecipitation (RIP), RNA pulldown and fluorescence in situ hybridization (FISH). Finally, we examined FOXF2 expression, ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related proteins in primary HESCs upon FOXF2 downregulation. Results FOXF2 was highly expressed in the endometrium of patients with IUA. Treatment of primary HESCs with 10 ng/ml TGF-β1 for 72 h was found to be most effective for developing an IUA cell model. FOXF2 regulated multiple downstream target genes, including collagen, vimentin (VIM) and cyclin D2/DK4, by ChIP-seq and ChIP-PCR. FOXF2 downregulation inhibited TGF-β1-mediated primary HESC fibrosis, including ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related protein expression. We identified lncRNA SNHG5 as an upstream gene that directly regulates FOXF2 by RIP-seq, qRT-PCR, WB and FISH. SNHG5 downregulation suppressed FOXF2 expression in the IUA cell model, resulting in synergistic repression of the Wnt/β-catenin pathway, thereby altering TGF-β1-mediated ECM aggregation in endometrial stromal cells ex vivo. Conclusions Regulation of the Wnt/β-catenin signalling pathway and ECM formation by si-SNHG5-FOXF2 effectively inhibited the profibrotic effect of TGF-β1 on primary HESCs. This finding can provide a molecular basis for antagonizing TGF-β1-mediated fibrosis in primary HESCs.

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