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.

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

2020 ◽  
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 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.

scholarly journals si-SNHG5-FOXF2 Inhibits TGF-β1-induced Fibrosis in Human Primary Endometrial Stromal Cells by Wnt/β-catenin Signaling Pathway

2020 ◽  
Author(s):  
Limin Liu ◽  
Guobin Chen ◽  
Taoliang Chen ◽  
Wenjuan Shi ◽  
Haiyan Hu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Signaling Pathway ◽  
Stromal Cells ◽  
Target Genes ◽  
Cell Model ◽  
Ex Vivo ◽  
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 si-SNHG5-FOXF2 can serve as a molecular mechanism for inhibition of IUA fibrosis ex vivo.Methods: FOXF2, TGF-β1 and collagens expression levels were measure by a Microarray sequencing analysis in three normal endometrium group and six IUA patients. We induced primary HESCs into MFs to develop an IUA cell model with various concentrations of TGF-β1 at various times. Downstream target genes for 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 signaling 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, EdU and CCK8 assays. We identified lncRNA SNHG5 as the upstream regulatory gene of FOXF2 through RNA immunoprecipitation (RIP), RNA-pulldown and FISH. Finally, we examined FOXF2 expression, ECM formation, cell proliferation and Wnt/ β-catenin signaling pathway-related proteins in primary HESCs with FOXF2 downregulation.Results: FOXF2 was highly expressed in endometrium from patients with IUA. Treatment of primary HESCs with 10ng / 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, 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 signaling pathway-related proteins expressions. We identified lncRNA SNHG5 as an upstream gene that directly regulated FOXF2 by RIP-seq, qRT-PCR, WB and FISH. SNHG5 downregulation suppressed FOXF2 expression in the IUA cell model, resulting in synergistic repression of Wnt/β-catenin signaling pathway activation and thereby altering TGF-β1-mediated ECM aggregation in endometrial stromal cells ex vivo. Conclusions: Regulation of the Wnt/β-catenin signaling pathway and ECM formation by si-SNHG5-FOXF2 effectively inhibited the profibrotic effect of TGF-β1 on primary HESCs. It can provide a molecular basis for antagonizing TGF-β1-mediated fibrosis in primary HESCs.

scholarly journals Activated Hippo/Yes-Associated Protein Pathway Promotes Cell Proliferation and Anti-apoptosis in Endometrial Stromal Cells of Endometriosis

2016 ◽  
Vol 101 (4) ◽  
pp. 1552-1561 ◽  
Author(s):  
Yong Song ◽  
Jing Fu ◽  
Min Zhou ◽  
Li Xiao ◽  
Xue Feng ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Signaling Pathway ◽  
Nude Mice ◽  
Stromal Cells ◽  
Target Genes ◽  
Critical Role ◽  
B Cell Lymphoma ◽  
Endometrial Stromal Cells ◽  
Proliferation And Apoptosis

Abstract Context: The imbalance in cell proliferation and apoptosis is considered an important role in the pathogenesis of endometriosis, but the exact mechanisms remains unclear. A newly established signaling pathway–Hippo/Yes-associated protein (YAP) pathway plays a critical role in the proliferation and apoptosis processes. However, studies focusing on Hippo/YAP pathway and endometriosis are lacking. Objective: The objective was to explore the function of the Hippo/YAP pathway in endometriosis. Setting and Design: The expression of YAP was first investigated in endometrium of women with or without endometriosis. The role of YAP in cell proliferation and apoptosis is identified by transfection of endometrial stromal cells (ESCs) in vitro, subsequent Verteporfin treatments in eutopic ESCs in vitro, and endometriosis animal model of nude mice in vivo. Results: Our results revealed that increased expression of YAP and decreased expression of p-YAP in ectopic and eutopic endometrium compared with normal endometrium. YAP knockdown in eutopic ESCs decreased cell proliferation and enhanced cell apoptosis companied with decreased expression of TEAD1, CTGF, and B-cell lymphoma/leukemia (BCL)-2; whereas overexpression of YAP resulted in increased proliferation and decreased apoptosis of normal ESCs with increased expression of TEAD1, CTGF, and BCL-2. By chromatin immunoprecipitation qPCR CTGF and BCL-2 were identified as directly downstream target genes of YAP-TEAD1 active complex. Eutopic ESCs treated with Verteporfin revealed decreased proliferation and enhanced apoptosis whereas in endometriosis animal models of nude mice treated with Verteporfin, the size of endometriotic lesions was significantly reduced. Conclusions: Our study suggests that the Hippo/YAP-signaling pathway plays a critical role in the pathogenesis of endometriosis and should present a novel therapeutic method against endometriosis.

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.

Upregulated Talin1 Synergistically Boosts β-estradiol-induced Proliferation and Pro-angiogenesis of Eutopic and Ectopic Endometrial Stromal Cells in Adenomyosis

2021 ◽  
Author(s):  
Yi-yi Wang ◽  
Hua Duan ◽  
Sha Wang ◽  
Yong-jun Quan ◽  
Jun-hua Huang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Intervention Group ◽  
Xenograft Model ◽  
Treated Group ◽  
Endometrial Stromal Cells ◽  
Growth And Survival ◽  
Human Carcinomas

Abstract Adenomyosis (ADS) is an estrogen-dependent gynecological disease with unspecified etiopathogenesis. Local hyperestrogenism may serve a central role in contributing the origin of ADS. Talin1 is mostly identified to be overexpressed and involved in the progression of numerous human carcinomas through mediating cell proliferation, adhesion and motility. Whether Talin1 exerts an oncogenic role in the development of ADS and presents an extra impact on the efficacy of estrogen, no relevant data are available yet. Here we demonstrated that the adenomyotic eutopic and ectopic endometrial stromal cells (ADS_Eu_ESC and ADS_Ec_ESC) treated with β-estradiol (β-E2) presented stronger proliferative and proangiogenetic capacities, accompanied by increased expression of PCNA, Ki67, VEGFB and ANGPTL4 proteins, compared with the controls. Meanwhile, these promoting effects were abrogated in the presence of Fulvestrant (ICI 182780, an estrogen-receptor antagonist). Aberrantly Upregulation of Talin1 mRNA and protein level was observed in ADS endometrial specimens and stromal cells. Through performing functional experiments in vitro, we further determined that merely overexpression of Talin1 (OV-Talin1) also enhanced ADS stromal cell proliferation and pro-angiogenesis, while the most pronounced facilitating effects were found in the co-intervention group of Talin1 overexpression plus β-E2 treatment. Results from the xenograft model showed that the hypodermic endometrial lesions from the co-treatment group with OV-Talin1 and β-E2 had the highest mean weight and volume, compared with that of individual OV-Talin1 or β-E2 treatment. The expression levels of PCNA, Ki67, VEGFB and ANGPTL4 in the lesions were correspondingly elevated most significantly in the co-treated group. Our findings unveiled that abnormally overexpressed Talin1 cooperated with E2 in stimulating ADS endometrial stromal cell proliferation and neovascularization, synergistically promoting the growth and survival of ectopic lesions. These results may be beneficial to provide a new insight for clarifying the pathogenesis of ADS.

Hypoxia and STAT3 signalling interactions regulate pro-inflammatory pathways in rheumatoid arthritis

2014 ◽  
Vol 74 (6) ◽  
pp. 1275-1283 ◽  
Author(s):  
Wei Gao ◽  
Jennifer McCormick ◽  
Mary Connolly ◽  
Emese Balogh ◽  
Douglas J Veale ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Target Genes ◽  
Ex Vivo ◽  
Notch 1 ◽  
Functional Link ◽  
Jak2 Inhibitor ◽  
Downstream Target ◽  
Inflammatory Pathways ◽  
And Migration ◽  
Spontaneous Secretion

ObjectiveTo examine the effect of hypoxia on Signal Transducer and Activator of Transcription 3 (STAT3)-induced pro-inflammatory pathways in rheumatoid arthritis (RA).MethodsDetection of phospho-STAT3 was assessed in RA synovial tissue and fibroblasts (RASFC) by immunohistology/immunofluorescence. Primary RASFCs and a normal synoviocyte cell line (K4IM) were cultured under hypoxic and normoxic conditions±Stat3-siRNA, HIF-siRNA or WP1066 (JAK2-inhibitor). HIF1α, p-STAT3, p-STAT1 and Notch-1IC protein expression were analysed by western blot. Functional mechanisms were quantified by invasion chamber, matrigel and migration assays. IL-6, IL-8, IL-10 and matrixmetalloproteinases (MMP)-3 were quantified by ELISA. Notch-1 receptor, its DLL-4 ligand and downstream target genes (hrt-1, hrt-2) were quantified by real-time PCR. The effect of WP1066 on spontaneous secretion of pro/anti-inflammatory cytokines and Notch signalling was examined in RA synovial explants ex vivo.Resultsp-STAT3 was increased in RA synovium compared with control (p<0.05). Hypoxia induced p-STAT3, p-STAT1 and HIF1α expression, an effect blocked by Stat3-siRNA and WP1066. Hypoxia-induced cell invasion, migration and cytokine production were inhibited by Stat3-siRNA (p<0.05) and WP1066 (p<0.05). While HIF1α siRNA inhibited hypoxia-induced p-STAT3 detection, Stat3-siRNA also inhibited hypoxia-induced HIF1α. Furthermore, hypoxia-induced Notch-1IC, DLL4, hrt-1 and -2 expression were significantly inhibited by WP1066 (p<0.05). Finally, in RA synovial explant cultures ex vivo, WP1066 decreased spontaneous secretion of IL-6, IL-8 and MMP3 (p<0.05), Notch-1 mRNA (p<0.05) and induced IL-10 (p<0.05).ConclusionsThis is the first study to provide evidence of a functional link between HIF1α, STAT3 and Notch-1 signalling in the regulation of pro-inflammatory mechanisms in RA, and further supports a role for STAT blockade in the treatment of RA.

TGF-β Receptor Kinase Inhibitor LY2109761 Reverses the Anti-Apoptotic Effects of TGF-β1 in Leukemic Cells Co-Cultured with Stromal Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2846-2846
Author(s):  
Yoko Tabe ◽  
Yuanyuan Xu ◽  
Teresa McQueen ◽  
Michael Andreeff ◽  
Marina Konopleva
Keyword(s):  
Cell Proliferation ◽  
Stromal Cells ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
U937 Cells ◽  
The Novel ◽  
Β Receptor ◽  
Tgf Β1 ◽  
Cells Cultured

Abstract Transforming growth factor β1 (TGF-β1) is an essential regulator of cell proliferation, survival, and apoptosis, depending on the cellular context. TGF-β1 is also known to affect cell-to-cell interactions between tumor and stromal cells through production of the extracellular matrix and stimulation of integrin receptors. We investigated the role of TGF-β1 in the survival of human leukemic cells growing in the context of bone marrow (BM) microenvironment, and the anti-leukemia effects of the novel TGF-β receptor inhibitor LY2109761. BM-derived mesenchymal stem cells (MSC) produced TGF-β in an autocrine fashion. Treatment with rhTGF-β1 (2ng/mL) inhibited the spontaneous and Ara-C-induced apoptosis in U937 cells (% AnnexinV(+), control 34.5±8.4; TGF-β1 18.4±4.5; Ara-C 88.6±3.0; Ara-C/TGF-β1 60.4±8.0, p=0.04). These effects were more prominent in U937 cells co-cultured with MSC (% AnnexinV(+), control 19.4±2.8; TGF-β1 3.5±1.0; Ara-C 69.0±3.6; Ara-C+TGF-β1 24.9±3.3; p=0.01). In U937 cells co-cultured with MSC, rhTGF-β1 conferred higher cell protective effects on leukemia cells attached to MSC than on floating cells. Conversely, the pro-survival effects of TGF-β1 were inhibited by 5mM LY2109761 (%AnnexinV(+); MSC(−), control 31.8±2.3, TGF-β1 19.5±3.0, LY 28.4±4.4, TGF-β1+LY 37.7±2.0; MSC(+), control 22.1±1.7, TGF-β1 7.8±0.9, LY16.1±2.6, TGF-β1+ LY 18.0±1.1, p<0.01). Similar results were obtained using TGF-β1 neutralizing antibody. TGF-β1 induced pro-survival phosphorylation of Akt in U937 cells cultured alone or co-cultured with MSC, which was abrogated by LY2109761. Further, rhTGF-β1 induced a moderate increase in C/EBPβ gene and LAP isoform (cell cycle arrest inducing) of C/EBPβ protein in U937 cells cultured without MSCs, while markedly upregulating C/EBPβ gene and protein, both LIP (cell proliferation inducing) and LAP isoforms, under MSCs co-culture condition, suggesting the novel role of C/EBPβ in TGF-β1-mediated U937 cell survival. In summary, these results indicate that TGF-β1-secreting BM stromal cells promote the survival of U937 leukemia cells via direct cell-to-cell interaction and promote chemoresistance of leukemia cells through the activation of Akt signaling and upregulation of C/EBPβ. The blockade of TGF-β signaling by LY2109761, which effectively inhibited the pro-survival signaling, may enhance the efficacy of chemotherapy against leukemia cells in the BM microenvironment.

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