scholarly journals MicroRNA 648 Targets ET-1 mRNA and Is Cotranscriptionally Regulated withMICAL3by PAX5

2014 ◽  
Vol 35 (3) ◽  
pp. 514-528 ◽  
Author(s):  
Chen Li ◽  
Caryn S. Gonsalves ◽  
Marthe-Sandrine Eiymo Mwa Mpollo ◽  
Punam Malik ◽  
Stanley M. Tahara ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Posttranscriptional Regulation ◽  
Hypoxia Inducible Factor ◽  
Positive Control ◽  
Luciferase Reporter ◽  
Lim Domain ◽  
Placenta Growth Factor ◽  
Reporter Assays ◽  
Potent Vasoconstrictor ◽  
Distal Promoter

Pulmonary hypertension (PHT) is associated with high mortality in sickle cell anemia (SCA). Previously, we showed that elevated levels of placenta growth factor (PlGF) in SCA patients correlate with increased levels of the potent vasoconstrictor endothelin-1 (ET-1) and PHT. Moreover, PlGF induced the expression of ET-1 via hypoxia-inducible factor 1α. Here, we show a novel example of ET-1 posttranscriptional regulation by PlGF via action of microRNA 648 (miR-648), which is subject to transcriptional coregulation with its host gene,MICAL3(microtubule-associated monooxygenase, calponin, and LIM domain containing 3gene). PlGF repressed expression of miR-648 in endothelial cells. Luciferase reporter assays using wild-type and mutant ET-1 3′ untranslated region (UTR) constructs, and transfection of miR-648 mimics showed that miR-648 targets the 3′ UTR of ET-1 mRNA. Since miR-648 is located in a 5′-proximal intron ofMICAL3, we examined which of three potential promoters was responsible for its expression. TheMICAL3distal promoter (P1) was the predominant promoter used for transcription of pre-miR-648, and it was under positive control by PAX5 (paired box protein 5) transcription factor, as demonstrated by the loss and gain of function of PAX5 activity, and chromatin immunoprecipitation analysis. These studies provide a novel link wherein PlGF-mediated downregulation of PAX5 attenuates miR-648 expression leading to increased ET-1 levels that are known to induce PHT in SCA.

scholarly journals Placenta growth factor augments endothelin-1 and endothelin-B receptor expression via hypoxia-inducible factor-1α

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 856-865 ◽  
Author(s):  
Nitin Patel ◽  
Caryn S. Gonsalves ◽  
Punam Malik ◽  
Vijay K. Kalra
Keyword(s):  
Growth Factor ◽  
Hypoxia Inducible Factor ◽  
Receptor Expression ◽  
Placenta Growth Factor ◽  
Promoter Regions ◽  
Endothelin B Receptor ◽  
Endothelin 1 ◽  
Hypoxia Inducible Factor 1Α ◽  
Endothelin B ◽  
Potent Vasoconstrictor

Abstract Pulmonary hypertension (PHT) develops in sickle cell disease (SCD) and is associated with high mortality. We previously showed that erythroid cells produce placenta growth factor (PlGF), which activates monocytes to induce proinflammatory cytochemokines, contributing to the baseline inflammation and severity in SCD. In this study, we observed that PlGF increased expression of endothelin-1 (ET-1) and endothelin-B receptor (ET-BR) from human pulmonary microvascular endothelial cells (HPMVECs) and monocytes, respectively. PlGF-mediated ET-1 and ET-BR expression occurred via activation of PI-3 kinase, reactive oxygen species and hypoxia inducible factor-1α (HIF-1α). PlGF increased binding of HIF-1α to the ET-1 and ET-BR promoters; this effect was abrogated with mutation of hypoxia response elements in the promoter regions and HIF-1α siRNA and confirmed by chromatin immunoprecipitation analysis. Furthermore, PlGF-mediated ET-1 release from HPMVECs and ET-BR expression in monocytes creates a PlGF–ET-1–ET-BR loop, leading to increased expression of MCP-1 and IL-8. Our studies show that PlGF-induced expression of the potent vasoconstrictor ET-1 and its cognate ET-BR receptor occur via activation of HIF-1α, independent of hypoxia. PlGF levels are intrinsically elevated from the increased red cell turnover in SCD and in other chronic anemia (eg, thalassemia) and may contribute to inflammation and PHT seen in these diseases.

scholarly journals Transcriptional Regulation of gga-miR-451 by AhR:Arnt in Mycoplasma gallisepticum (HS Strain) Infection

2019 ◽  
Vol 20 (12) ◽  
pp. 3087 ◽  
Author(s):  
Yabo Zhao ◽  
Yali Fu ◽  
Yingfei Sun ◽  
Mengyun Zou ◽  
Xiuli Peng
Keyword(s):  
Aryl Hydrocarbon Receptor ◽  
Chromatin Immunoprecipitation ◽  
Regulatory Region ◽  
Mycoplasma Gallisepticum ◽  
Luciferase Reporter ◽  
Binding Motif ◽  
Aryl Hydrocarbon ◽  
Transcriptional Regulatory ◽  
Reporter Assays ◽  
Transcriptional Regulatory Region

MicroRNAs (miRNAs) have been determined to be important regulators for pathogenic microorganism infection. However, it is largely unclear how miRNAs are triggered during pathogen infection. We previously reported that the up-regulation of gga-miR-451 negatively regulates the Mycoplasma gallisepticum (MG)-induced production of inflammatory cytokines via targeting tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein zeta (YWHAZ). The aim of this study was to investigate the mechanism regulating gga-miR-451 in MG infection in chickens. Analysis of gga-miR-451 precursor, pri-miR-451, and pre-miR-451 indicated that the regulation occurred transcriptionally. We also identified the transcriptional regulatory region of gga-miR-451 that contained consensus-binding motif for aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (Arnt) complex, which is known as the transcription factor that regulates gene expression. Luciferase reporter assays combined with chromatin immunoprecipitation (ChIP) demonstrated that AhR:Arnt bound directly to the promoter elements of gga-miR-451, which were responsible for gga-miR-451 transcription in the context of MG infection. Furthermore, upregulation of AhR:Arnt significantly induced gga-miR-451 and inhibited YWHAZ expression, suggesting that AhR:Arnt may play an anti-inflammatory role in MG infection. This discovery suggests that induced gga-miR-451 expression is modulated by AhR:Arnt in response to MG infection.

scholarly journals Estrogen-Related Receptor-α Promotes Pancreatic Cancer Progression by Enhancing the Transcription of PAI1 and Activating the MEK/ERK Signaling Pathway

2020 ◽  
Author(s):  
Shi-lei Liu ◽  
Xiang-song Wu ◽  
Feng-nan Li ◽  
Wen-yan Yao ◽  
Zi-you Wu ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
Rna Sequencing ◽  
Chromatin Immunoprecipitation ◽  
Erk Signaling ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Erk Pathway ◽  
Reporter Assays ◽  
Estrogen Related Receptor

Abstract Background: Estrogen-related receptor alpha (ERRα), an orphan nuclear receptor, was reported to be highly associated with the progression and tumorigenesis of several human malignancies. However, the biological role and underlying molecular mechanisms of ERRα in pancreatic cancer (PC) remain unknown.Methods: The expression of ERRα in PC tissues was determined by qRT-PCR and immunohistochemistry. A series of in vitro and in vivo assays were performed to investigate the function of ERRα and Plasminogen activator inhibitor 1 (PAI1) in tumorigenesis in PC cells. The relationship between ERRα and PAI1 was identified by RNA sequencing, Chromatin immunoprecipitation and dual-luciferase reporter assays. The effects of ERRα on the MEK/ERK signaling pathway were determined by western blotting and rescue assays using ERK inhibitor GDC-0994.Results: ERRα was significantly overexpressed in PC tissues and cell lines. Its high expression was correlated with tumor size, distant metastasis, TNM stage, tumor differentiation and poor prognosis of PC. Subsequent functional assays showed that ERRα promoted PC cell proliferation, tumor growth, as well as migration and invasion via activating the epithelial-mesenchymal transition. In addition, knockdown of ERRα induced apoptosis and G0/G1 cell cycle arrest in PC cells. PAI1 was identified by RNA sequencing, knockdown of which could suppress the cell proliferation, migration and invasion that promoted by ERRα overexpression. Further mechanistic investigation using chromatin immunoprecipitation and dual-luciferase reporter assays revealed that ERRα could bind to the PAI1 promoter region and transcriptionally enhance PAI1 expression. Moreover, our data indicated that ERRα played its oncogenic role in PC via activating the MEK/ERK pathway.Conclusions: Our study demonstrates that ERRα promotes PC progression by enhancing the transcription of PAI1 and activation of the MEK/ERK pathway, pointing to ERRα as a novel diagnostic and therapeutic target for PC.

scholarly journals Components from the Steamed Leaves of Acanthopanax koreanum and their Effects on PPAR Activity in HepG2 Cells

2011 ◽  
Vol 6 (9) ◽  
pp. 1934578X1100600
Author(s):  
Jeong Ah Kim ◽  
Seok Bean Song ◽  
Seo Young Yang ◽  
Young Ho Kim
Keyword(s):  
Mass Spectrometry ◽  
Biological Effects ◽  
Positive Control ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Acanthopanax Koreanum ◽  
Specificity And Sensitivity ◽  
Reporter Assays ◽  
C Nmr

Three ent-kaurane diterpenes (1-3), four lupane-triterpene glycosides (4-7), and an oleanane-triterpene glycoside (8) were isolated from the ethyl acetate and water extracts of the steamed leaves of Acanthopanax koreanum using a combination of various column chromatographies. The structures of the isolates were determined by 1H-, 13C-NMR spectroscopy and mass spectrometry. To investigate the biological effects of the eight compounds (1-8) on peroxisome proliferator-activated receptor gamma (PPARγ), luciferase reporter assays were used. Among the tested compounds, ent-kaur-16-en-19-oic acid (1), 16α-hydroxy- ent-kauran-19-oic acid (2), and 17-hydroxy- ent-kaur-15-en-19-oic acid (3) showed considerable effects on PPARγ activity, compared with the positive control, troglitazone. To evaluate specificity and sensitivity of the active compounds (1-3) in the regulation of transactivation of PPARs, Gal4-PPARs-LBD luciferase reporter assays were examined. In this study, the three ent-kaurane diterpenes (1-3) were found to up-regulate PPARβ/δ and PPARγ activities, whereas they did not activate PPARα activity.

scholarly journals RPL34 regulates osteosarcoma cells proliferation through c-Myc/RPL34 signaling axis

2021 ◽  
Author(s):  
Piwei Huang ◽  
Minghui Wei ◽  
Shufan Ji ◽  
Mitra Fowdur ◽  
maolin he
Keyword(s):  
Ribosomal Protein ◽  
Chromatin Immunoprecipitation ◽  
Cell Cycle Progression ◽  
Luciferase Reporter ◽  
Osteosarcoma Cells ◽  
Cycle Progression ◽  
E Box ◽  
Signaling Axis ◽  
New Ideas ◽  
Reporter Assays

Abstract BackgroundRibosomal protein L34 (RPL34) is a member of the L34E ribosomal protein family containing zinc finger domains. This protein plays a key role in regulating the apoptosis, cell cycle progression and proliferation of various cancer including osteosarcoma (OS). The purpose of this study is to clarify the expression of RPL34 in osteosarcoma cells and its molecular mechanism of regulating osteosarcoma cells. MethodsThe expression levels of c-Myc and RPL34 were detected by qRT-PCR and Western blot. Luciferase reporter assays and chromatin immunoprecipitation (ChIP) were used to analyse the binding site of c-Myc and RPL34. ResultsThe results showed that c-Myc binds to the E-box region in the RPL34 promoter to regulate RPL34 expression. The results indicated that RPL34 regulates osteosarcoma cells proliferation through c-Myc/RPL34 signaling axis. This research may provide new ideas for targeted therapy of OS. Conclusion RPL34 regulates osteosarcoma cells proliferation through c-Myc/RPL34 signaling axis.

scholarly journals Sequential Activation of Hypoxia-Inducible Factor 1 and Specificity Protein 1 is Required for Hypoxia-Induced Transcriptional Stimulation of Abcc8

2011 ◽  
Vol 32 (3) ◽  
pp. 525-536 ◽  
Author(s):  
Seung Kyoon Woo ◽  
Min Seong Kwon ◽  
Zhihua Geng ◽  
Zheng Chen ◽  
Alexander Ivanov ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Binding Sites ◽  
Hypoxia Inducible Factor ◽  
Luciferase Reporter ◽  
Hypoxia Inducible Factor 1 ◽  
Specificity Protein 1 ◽  
Reporter Assays ◽  
Sequential Activation ◽  
Specificity Protein ◽  
Stimulation Of

Cerebral ischemia causes increased transcription of sulfonylurea receptor 1 (SUR1), which forms SUR1-regulated NC(Ca-ATP) channels linked to cerebral edema. We tested the hypothesis that hypoxia is an initial signal that stimulates transcription of Abcc8, the gene encoding SUR1, via activation of hypoxia-inducible factor 1 (HIF1). In the brain microvascular endothelial cells, hypoxia increased SUR1 abundance and expression of functional SUR1-regulated NC(Ca-ATP) channels. Luciferase reporter activity driven by the Abcc8 promoter was increased by hypoxia and by coexpression of HIF1α. Surprisingly, a series of luciferase reporter assays studying the Abcc8 promoter revealed that binding sites for specificity protein 1 (Sp1), but not for HIF, were required for stimulation of Abcc8 transcription by HIF1α. Luciferase reporter assays studying Sp1 promoters of three species, and chromatin immunoprecipitation analysis in rats after cerebral ischemia, indicated that HIF binds to HIF-binding sites on the Sp1 promoter to stimulate transcription of the Sp1 gene. We conclude that sequential activation of two transcription factors, HIF and Sp1, is required to stimulate transcription of Abcc8 following cerebral ischemia. Sequential gene activation in cerebral ischemia provides a plausible molecular explanation for the prolonged treatment window observed for inhibition of the end-target gene product, SUR1, by glibenclamide.

scholarly journals HIF-1α-induced expression of m6A reader YTHDF1 drives hypoxia-induced autophagy and malignancy of hepatocellular carcinoma by promoting ATG2A and ATG14 translation

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Qing Li ◽  
Yong Ni ◽  
Liren Zhang ◽  
Runqiu Jiang ◽  
Jing Xu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Chromatin Immunoprecipitation ◽  
Cox Regression ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Cox Regression Analysis ◽  
Polysome Profiling ◽  
Rna Immunoprecipitation ◽  
Reporter Assays

AbstractN6-methyladenosine (m6A), and its reader protein YTHDF1, play a pivotal role in human tumorigenesis by affecting nearly every stage of RNA metabolism. Autophagy activation is one of the ways by which cancer cells survive hypoxia. However, the possible involvement of m6A modification of mRNA in hypoxia-induced autophagy was unexplored in human hepatocellular carcinoma (HCC). In this study, specific variations in YTHDF1 expression were detected in YTHDF1-overexpressing, -knockout, and -knockdown HCC cells, HCC organoids, and HCC patient-derived xenograft (PDX) murine models. YTHDF1 expression and hypoxia-induced autophagy were significantly correlated in vitro; significant overexpression of YTHDF1 in HCC tissues was associated with poor prognosis. Multivariate cox regression analysis identified YTHDF1 expression as an independent prognostic factor in patients with HCC. Multiple HCC models confirmed that YTHDF1 deficiency inhibited HCC autophagy, growth, and metastasis. Luciferase reporter assays and chromatin immunoprecipitation demonstrated that HIF-1α regulated YTHDF1 transcription by directly binding to its promoter region under hypoxia. The results of methylated RNA immunoprecipitation sequencing, proteomics, and polysome profiling indicated that YTHDF1 contributed to the translation of autophagy-related genes ATG2A and ATG14 by binding to m6A-modified ATG2A and ATG14 mRNA, thus facilitating autophagy and autophagy-related malignancy of HCC. Taken together, HIF-1α-induced YTHDF1 expression was associated with hypoxia-induced autophagy and autophagy-related HCC progression via promoting translation of autophagy-related genes ATG2A and ATG14 in a m6A-dependent manner. Our findings suggest that YTHDF1 is a potential prognostic biomarker and therapeutic target for patients with HCC.

VEGF-mediated angiogenesis in retinopathy of prematurity is co-regulated by miR-17-5p and miR-20a-5p

2021 ◽  
pp. 1-10
Author(s):  
Yan Guo ◽  
Fen Du ◽  
Yi-Lan Tan ◽  
Jun Luo ◽  
Dan Xiong ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Retinopathy Of Prematurity ◽  
Hypoxia Inducible Factor ◽  
Luciferase Reporter ◽  
Oxygen Induced Retinopathy ◽  
Rna Immunoprecipitation ◽  
Reporter Assays ◽  
Retinal Angiogenesis ◽  
Labeled Rna ◽  
Endothelial Growth Factor

The microRNAs miR-17-5p and miR-20a-5p play important roles on angiogenesis; however, it is arguable whether they regulate the formation of retinal blood vessels in retinopathy of prematurity (ROP). We used a mouse model of oxygen-induced retinopathy (OIR) to simulate the development of retinas in mice suffering from ROP, and the expression levels of miR-20a-5p, miR-17-5p, hypoxia-inducible factor 1-alpha (HIF-1α), and vascular endothelial growth factor (VEGF) were measured by RT-qPCR and Western blotting. Cell proliferation, apoptosis, and angiogenesis in the OIR model mice were measured using MTT assays, flow cytometry, and Matrigel assays, respectively. The interaction between HIF-1α/VEGF and miR-20a-5p/miR-17-5p were further validated using dual-luciferase reporter assays, biotin-labeled RNA-pulldown, and RNA immunoprecipitation (RIP) assays. In our OIR model, retinal angiogenesis in the mice was associated with down-regulation of miR-20a-5p and miR-17-5p, as well as up-regulation of HIF-1α and VEGF. In addition, the miR-20a-5p and miR-17-5p inhibited cell proliferation and angiogenesis through regulating HIF-1α and VEGF in the retinal cells of the OIR model mice. Moreover, it was found that miR-20a-5p and miR-17-5p bind to HIF-1α and VEGF at the 3′UTR, and there was a combined effect between miR-20a-5p and miR-17-5p on the regulation of HIF-1α and VEGF. It is worth noting that miR-17-5p and miR-20a-5p can preferentially regulate HIF-1α, then act on VEGF, thereby affecting the angiogenesis associated with ROP.

scholarly journals Oxygen impairs oligodendroglial development via oxidative stress and reduced expression of HIF-1α

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Christina Brill ◽  
Till Scheuer ◽  
Christoph Bührer ◽  
Stefanie Endesfelder ◽  
Thomas Schmitz
Keyword(s):  
Oxidative Stress ◽  
Hypoxia Inducible Factor ◽  
Precursor Cell ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Low Oxygen ◽  
Hypoxia Inducible Factor 1 ◽  
Reporter Assays ◽  
Induced Changes ◽  
Cells Cultured

Abstract The premature increase of oxygen tension may contribute to oligodendroglial precursor cell (OPC) damage in preterm infants. Fetal OPCs are exposed to low oxygen tissue tensions not matched when cells are cultured in room air. Maturation (A2B5, O4, O1, MBP, CNP, arborization), oxidative stress (nitrotyrosine Western blot, NRF2 and SOD2 expression), apoptosis (TUNEL), proliferation (Ki67), and expression of transcription factors regulated by Hypoxia-Inducible-Factor-1-alpha (Hif-1α) expressed in OPCs (Olig1, Olig2, Sox9, Sox10) were assessed in rat OPCs and OLN93 cells cultured at 5% O2 and 21% O2. Influences of Hif-1α were investigated by Hif-1α luciferase reporter assays and Hif-1α-knockdown experiments. At 21% O2, cell proliferation was decreased and process arborization of OPCs was reduced. Expression of MBP, CNP, Olig1, Sox9 and Sox10 was lower at 21% O2, while Nrf2, SOD2, nitrotyrosine were increased. Apoptosis was unchanged. Luciferease reporter assay in OLN93 cells indicated increased Hif-1α activity at 5% O2. In OLN93 cells at 5% O2, Hif-1α knockdown decreased the expression of MBP and CNP, similar to that observed at 21% O2. These data indicate that culturing OPCs at 21% O2 negatively affects development and maturation. Both enhanced oxidative stress and reduced expression of Hif-1α-regulated genes contribute to these hyperoxia-induced changes.

Abstract 13494: Lncrna Khps1 Regulates Hif-1a-dependent Pulmonary Vascular Remodeling by Targeting Mir-1, Sphk1 and S1pr2

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Yang Bai ◽  
Angelia Lockett ◽  
Marta T Gomes ◽  
Nicole Jones ◽  
Alan de Brito Carneiro ◽  
...  
Keyword(s):  
Vascular Remodeling ◽  
Noncoding Rna ◽  
Hypoxia Inducible Factor ◽  
In Silico Analysis ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Pulmonary Vascular Remodeling ◽  
Expression Levels ◽  
Increased Risk ◽  
Reporter Assays

Rationale: Pulmonary vascular remodeling is common in pulmonary arterial hypertension (PAH) and is associated with increased risk of right ventricular failure and death. We have previously reported that sphingosine kinase 1 (Sphk1), sphingosine 1 phosphate receptor 2 (S1PR2), microRNA-1-3p (miR-1-3p) and hypoxia-inducible factor-1α (HIF-1α) are involved in the regulation of pulmonary vascular remodeling and the development of PAH. Recently, it has been reported that the long noncoding RNA (LncRNA) Khps1 is required for Sphk1 expression and cell proliferation. However, whether Khps1 plays a role in PAH is unclear. Here, we tested the hypothesis that Khps1 has an impact on pulmonary vascular remodeling in PAH by regulating Sphk1/S1PR2/HIF-1α signaling pathway. Methods and Results: Using qRT-PCR, our data showed that Khps1 expression levels were up-regulated in pulmonary artery smooth muscle cells (PASMCs) from PAH patients. Increased Khps1 expression levels were also observed in human PASMCs exposed to hypoxia and in lungs obtained from monocrotaline (MCT)-treated rats or hypoxia exposed mice. Luciferase reporter assays showed that Khps1 expression is dependent on E2F1-mediated promoter activation. In silico analysis and luciferase reporter assays also suggested that Khps1 can directly bind to miR-1-3p, suggesting that Khps1 functions as a competing endogenous RNA. Similar studies also showed that miR-1-3p regulates expression of Sphk1 and S1PR2 by binding to their 3’UTR regions. Concurrent E2F1 overexpression and khps1 silencing resulted in a decrease in Sphk1 and S1PR2 expression levels, reduced HIF-1α stabilization and suppressed PASMCs proliferation. Finally, over expression of Sphk1 increased HIF-1α and that in turn increased E2F1 expression in human PASMCs. Conclusion: These data reveal a novel regulatory loop between E2F1, Khps1, miR-1-3p and Sphk1/S1PR2 in the regulation of PASMC proliferation and pulmonary vascular remodeling in PAH. These data also suggest Khps1 regulates pulmonary vascular remodeling in a HIF-1α-dependent manner through targeting Sphk1 and S1PR2.

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