scholarly journals Etv5, an ETS transcription factor, is expressed in granulosa and cumulus cells and serves as a transcriptional regulator of the cyclooxygenase-2

2008 ◽  
Vol 198 (2) ◽  
pp. 281-290 ◽  
Author(s):  
Jinwon Eo ◽  
Kyuyong Han ◽  
Kenneth M Murphy ◽  
Haengseok Song ◽  
Hyunjung Jade Lim
Keyword(s):  
Transcriptional Activity ◽  
Cumulus Cells ◽  
Cyclooxygenase 2 ◽  
Luciferase Reporter ◽  
Ets Transcription Factors ◽  
Cellular Events ◽  
Reporter Assays ◽  
Ets Transcription Factor ◽  
Cell Niche ◽  
Rate Limiting

Etv4, Etv1, and Etv5 are members of Etv4 subfamily of E26 transformation-specific (Ets) transcription factors that are known to influence a host of biological processes. We previously showed that Etv5, expressed in Sertoli cells, plays a crucial role in maintaining spermatogonial stem cell niche in the mouse testis. However, it is not yet known whether Etv4 family members are expressed in the ovary or play any role in ovarian functions. Here, we show that Etv5 and Etv4 are expressed in mouse ovaries in granulosa and cumulus cells during folliculogenesis. Both Etv5 and Etv4 mRNAs are also detected in cumulus–oocyte complexes (COCs) and denuded oocytes. Notably, Etv4 is highly expressed in the cumulus cells of ovulated COCs at 16-h post-human chorionic gonadotropin. Cyclooxygenase-2 (PTGS2), a rate-limiting enzyme for prostaglandin synthesis, is critical for oocyte maturation and ovulation. Since several putative Ets-binding sites are present in the PTGS2 promoter, we examined whether Etv5 influences Ptgs2 transcriptional activity. Indeed, we found that addition of Etv5 increases the transcriptional activity of the 3.2-kb mouse Ptgs2 promoter by 2.5-fold in luciferase reporter assays. Collectively, the results show that Etv4 and Etv5 are expressed in granulosa and cumulus cells during folliculogenesis and ovulation, suggesting that they influence cellular events in the ovary by regulating downstream genes such as Ptgs2.

scholarly journals Cross-talk between SIM2s and NFkB regulates cyclooxygenase 2 expression in breast cancer

2019 ◽  
Author(s):  
Garhett Wyatt ◽  
Chloe Young ◽  
Lyndsey Crump ◽  
Veronica Wessells ◽  
Tanya Gustafson ◽  
...  
Keyword(s):  
Breast Cancer ◽  
The United States ◽  
Cyclooxygenase 2 ◽  
Luciferase Reporter ◽  
Signaling Proteins ◽  
Dependent Manner ◽  
Human Breast Carcinomas ◽  
Reporter Assays ◽  
Cox 2

AbstractBackgroundBreast cancer is a leading cause of cancer-related death for women in the United States. Thus, there a need to investigate novel prognostic markers and therapeutic strategies. Inflammation raises challenges to both treating and preventing the spread of breast cancer. Specifically, the nuclear factor kappa b (NFkB) pathway contributes to cancer progression by stimulating proliferation and preventing apoptosis. One target gene of this pathway is PTGS2, the gene that encodes for cyclooxygenase 2 (COX-2), which is upregulated in 40% of human breast carcinomas. COX-2 is an enzyme involved in inflammation. Here we investigate the effect of Singleminded 2s, a transcriptional tumor suppressor that is implicated in inhibition of tumor growth and metastasis, in regulating NFkB and COX-2.MethodsWe utilized in vitro reporter assays, immunoblot analyses, qPCR and immunohistochemical analysis to dissect the relationship between NFκB, SIM2s, and COX-2. Furthermore, we utilized COX-2 targeting strategies to determine tumor suppressive activities.ResultsOur results reveal that SIM2s attenuates the activation of a NFκB via luciferase reporter assays. Furthermore, immunostaining of lysates from breast cancer cells over expressing SIM2s showed reduction in various NFκB signaling proteins, whereas knockdown of SIM2 revealed increases in the same NFκB signaling proteins. Additionally, by increasing NFκB translocation to the nucleus in DCIS.COM cells, we show that NFκB signaling can act in a reciprocal manner to decrease expression of SIM2s. Likewise, suppressing NFκB translocation in DCIS.COM cells increases SIM2s expression. We also found that NFκB/p65 represses SIM2 in via dose-dependent manner and when NFκB is suppressed the effect on the SIM2 is negated. Additionally, our CHIP analysis confirms that NFκB/p65 binds directly to SIM2 promoter site and that the NFκB sites in the SIM2 promoter are required for NFkB-mediated suppression of SIM2s. Finally, over expression of SIM2s decreases PTGS2 in vitro and COX-2 staining in vivo while decreasing PTGS2 and/or Cox-2 activity results in re-expression of SIM2. Our findings identify a novel role for SIM2s in NFκB signaling and COX-2 expression.ConclusionsThese findings provide evidence for a mechanism where SIM2s may represses COX-2 expression to provide an overall better prognosis for breast cancer patients.

scholarly journals Identification of the Repressive Domain of the Negative Circadian Clock Component CHRONO

2019 ◽  
Author(s):  
Yu Yang ◽  
Ning Li ◽  
Jiameng Qiu ◽  
Honghua Ge ◽  
Ximing Qin
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activity ◽  
Structural Information ◽  
Luciferase Reporter ◽  
Circadian Cycle ◽  
Information Analysis ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Clock Component ◽  
Reporter Assays

AbstractCircadian rhythm is an endogenous, self-sustainable oscillation that participates in regulating organisms’ physiological activities. Key to this oscillation is a negative feedback by main clock components Periods and Cryptochromes that repress transcriptional activity of BMAL1/CLOCK complexes. In addition, a novel repressor, CHRONO, has been identified recently, but details of CHRONO’s function during repressing the circadian cycle remain unclear. Here we report that a domain of CHRONO mainly composed of α-helixes is critical to repression through the exploitation of protein-protein interactions according to luciferase reporter assays, co-immunoprecipitation, immunofluorescence, genome editing and structural information analysis via circular dichroism spectroscopy. This repression is fulfilled by interactions between CHRONO with a region on the BMAL1 C-terminus where Cryptochrome and CBP bind. Our results indicate that CHRONO and PER differentially function as BMALA/CLOCK-dependent repressors. We further suggest a repression model of how PER, CRY and CHRONO proteins associate with BMAL1, respectively.

scholarly journals Identification of the Repressive Domain of the Negative Circadian Clock Component CHRONO

2020 ◽  
Vol 21 (7) ◽  
pp. 2469
Author(s):  
Yu Yang ◽  
Ning Li ◽  
Jiameng Qiu ◽  
Honghua Ge ◽  
Ximing Qin
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activity ◽  
Structural Information ◽  
Luciferase Reporter ◽  
Circadian Cycle ◽  
Information Analysis ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Clock Component ◽  
Reporter Assays

Circadian rhythm is an endogenous, self-sustainable oscillation that participates in regulating organisms’ physiological activities. Key to this oscillation is a negative feedback by the main clock components Periods and Cryptochromes that repress the transcriptional activity of BMAL1/CLOCK (defined in the Abbreviations) complexes. In addition, a novel repressor, CHRONO, has been identified recently, but details of CHRONO’s function during repressing the circadian cycle remain unclear. Here we report that a domain of CHRONO mainly composed of α-helixes is critical to repression through the exploitation of protein–protein interactions according to luciferase reporter assays, co-immunoprecipitation, immunofluorescence, genome editing, and structural information analysis via circular dichroism spectroscopy. This repression is fulfilled by interactions between CHRONO and a region on the C-terminus of BMAL1 where Cryptochrome and CBP (defined in the Abbreviations) bind. Our resultsindicate that CHRONO and PER differentially function as BMAL1/CLOCK-dependent repressors. Besides, the N-terminus of CHRONO is important for its nuclear localization. We further develop a repression model of how PER, CRY, and CHRONO proteins associate with BMAL1, respectively.

scholarly journals Structural Features and Transcriptional Activity of Chicken PPARs (α,β, andγ)

PPAR Research ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Ichiro Takada ◽  
Mime Kobayashi
Keyword(s):  
Lipid Metabolism ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Ppar Gamma ◽  
Structural Features ◽  
Luciferase Reporter ◽  
Ligand Selectivity ◽  
Adult Bovine Serum ◽  
Reporter Assays ◽  
Free Media

While an understanding of lipid metabolism in chickens is critical for a further improvement of food production, there are few studies concerning differences in lipid metabolism mechanisms between chickens and other species at a molecular level. Chickens have three PPAR gene subtypes (α,β, andγ) that function differently from those present in humans and mice. The chicken PPAR-gamma (cPPARγ) gene is shorter than that in humans and lacks aγ2 isoform. Moreover, in serum-free media, cPPARγshows high transcriptional activity without exogenous ligands. Luciferase reporter assays were used to examine the effect of sera on cPPAR transcriptional activities and showed that adult bovine serum and chicken serum highly activate cPPARαandβfunctions. Moreover, we found that bezafibrate induces the transactivation function of cPPARβ, but not human PPARδ(human PPARβortholog). This ligand selectivity relies on one amino acid residue (chicken: Val419, human: Met444). These results show the possibilities for unique functions of cPPARs on chicken-specific lipid glucose metabolism. As such, a better understanding of the molecular mechanisms of lipid metabolism in chickens could result in higher productivity for the poultry industry.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

scholarly journals TEAD4 modulated LncRNA MNX1-AS1 contributes to gastric cancer progression partly through suppressing BTG2 and activating BCL2

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
You Shuai ◽  
Zhonghua Ma ◽  
Weitao Liu ◽  
Tao Yu ◽  
Changsheng Yan ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Mechanistic Model ◽  
Ectopic Expression ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Tissue Samples ◽  
Reporter Assays

Abstract Background Gastric cancer (GC) is the third leading cause of cancer-related mortality globally. Long noncoding RNAs (lncRNAs) are dysregulated in obvious malignancies including GC and exploring the regulatory mechanisms underlying their expression is an attractive research area. However, these molecular mechanisms require further clarification, especially upstream mechanisms. Methods LncRNA MNX1-AS1 expression in GC tissue samples was investigated via microarray analysis and further determined in a cohort of GC tissues via quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. Cell proliferation and flow cytometry assays were performed to confirm the roles of MNX1-AS1 in GC proliferation, cell cycle regulation, and apoptosis. The influence of MNX1-AS1 on GC cell migration and invasion was explored with Transwell assays. A xenograft tumour model was established to verify the effects of MNX1-AS1 on in vivo tumourigenesis. The TEAD4-involved upstream regulatory mechanism of MNX1-AS1 was explored through ChIP and luciferase reporter assays. The mechanistic model of MNX1-AS1 in regulating gene expression was further detected by subcellular fractionation, FISH, RIP, ChIP and luciferase reporter assays. Results It was found that MNX1-AS1 displayed obvious upregulation in GC tissue samples and cell lines, and ectopic expression of MNX1-AS1 predicted poor clinical outcomes for patients with GC. Overexpressed MNX1-AS1 expression promoted proliferation, migration and invasion of GC cells markedly, whereas decreased MNX1-AS1 expression elicited the opposite effects. Consistent with the in vitro results, MNX1-AS1 depletion effectively inhibited the growth of xenograft tumour in vivo. Mechanistically, TEAD4 directly bound the promoter region of MNX1-AS1 and stimulated the transcription of MNX1-AS1. Furthermore, MNX1-AS1 can sponge miR-6785-5p to upregulate the expression of BCL2 in GC cells. Meanwhile, MNX1-AS1 suppressed the transcription of BTG2 by recruiting polycomb repressive complex 2 to BTG2 promoter regions. Conclusions Our findings demonstrate that MNX1-AS1 may be able to serve as a prognostic indicator in GC patients and that TEAD4-activatd MNX1-AS1 can promote GC progression through EZH2/BTG2 and miR-6785-5p/BCL2 axes, implicating it as a novel and potent target for the treatment of GC.

scholarly journals Circular RNA mmu_circ_0005019 inhibits fibrosis of cardiac fibroblasts and reverses electrical remodeling of cardiomyocytes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Na Wu ◽  
Chengying Li ◽  
Bin Xu ◽  
Ying Xiang ◽  
Xiaoyue Jia ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cardiac Fibroblasts ◽  
Circular Rna ◽  
Protective Role ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Candidate Target ◽  
Paired Control ◽  
Reporter Assays ◽  
And Migration

Abstract Background Circular RNA (circRNA) have been reported to play important roles in cardiovascular diseases including myocardial infarction and heart failure. However, the role of circRNA in atrial fibrillation (AF) has rarely been investigated. We recently found a circRNA hsa_circ_0099734 was significantly differentially expressed in the AF patients atrial tissues compared to paired control. We aim to investigate the functional role and molecular mechanisms of mmu_circ_0005019 which is the homologous circRNA in mice of hsa_circ_0099734 in AF. Methods In order to investigate the effect of mmu_circ_0005019 on the proliferation, migration, differentiation into myofibroblasts and expression of collagen of cardiac fibroblasts, and the effect of mmu_circ_0005019 on the apoptosis and expression of Ito, INA and SK3 of cardiomyocytes, gain- and loss-of-function of cell models were established in mice cardiac fibroblasts and HL-1 atrial myocytes. Dual-luciferase reporter assays and RIP were performed to verify the binding effects between mmu_circ_0005019 and its target microRNA (miRNA). Results In cardiac fibroblasts, mmu_circ_0005019 showed inhibitory effects on cell proliferation and migration. In cardiomyocytes, overexpression of mmu_circ_0005019 promoted Kcnd1, Scn5a and Kcnn3 expression. Knockdown of mmu_circ_0005019 inhibited the expression of Kcnd1, Kcnd3, Scn5a and Kcnn3. Mechanistically, mmu_circ_0005019 exerted biological functions by acting as a miR-499-5p sponge to regulate the expression of its target gene Kcnn3. Conclusions Our findings highlight mmu_circ_0005019 played a protective role in AF development and might serve as an attractive candidate target for AF treatment.

LncRNA LINC00858 enhances cervical cancer cell growth through miR-3064-5p/ VMA21 axis

2021 ◽  
pp. 1-11
Author(s):  
Min Wei ◽  
Youguo Chen ◽  
Wensheng Du
Keyword(s):  
Cervical Cancer ◽  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Luciferase Reporter ◽  
Cancer Cell Growth ◽  
Protein Coding ◽  
Gynecological Malignancy ◽  
Promising Therapeutic Target ◽  
Proliferation And Apoptosis ◽  
Reporter Assays

BACKGROUND: Cervical cancer (CC) is the most common form of gynecological malignancy. Long intergenic non-protein coding RNA 858 (LINC00858) has been identified to participate in multiple cancers. However, the role and mechanism of LINC00858 in CC cells are still elusive. AIM: The aim of this study is to explore the biological functions and mechanisms of LINC00858 in CC cells. METHODS: RT-qPCR analysis was used to examine the expression of LINC00858 in CC cells. EdU and colony formation assay were utilized to assess cell proliferation. TUNEL assay and flow cytometry assay were conducted to assess cell apoptosis. The mechanism regarding LINC00858 was certified through RNA pull down, RIP and luciferase reporter assays. RESULTS: The up-regulated LINC00858 was detected in CC cells. Reduction of LINC00858 effectively subdued CC cells proliferation and stimulated cell apoptosis. LINC00858 was determined to bind with miR-3064-5p and up-regulate VMA21 in CC cells. In rescue assays, miR-3064-5p down-regulation and VMA21 up-regulation were able to counteract the effect caused by LINC00858 decrease on CC cell proliferation and apoptosis. CONCLUSION: LINC00858 enhances cell proliferation, while restraining cell apoptosis in CC through targeting miR-3064-5p/VMA21 axis, implying that LINC00858 may serve as a promising therapeutic target for CC.

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