scholarly journals Hypoxic up-regulation of erythroid 5-aminolevulinate synthase

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 348-350 ◽  
Author(s):  
Thomas Hofer ◽  
Roland H. Wenger ◽  
Marianne F. Kramer ◽  
Gloria C. Ferreira ◽  
Max Gassmann
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Hypoxia Inducible Factor ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Binding Assays ◽  
Luciferase Reporter Gene ◽  
Aminolevulinate Synthase ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

Abstract The erythroid-specific isoform of 5-aminolevulinate synthase (ALAS2) catalyzes the rate-limiting step in heme biosynthesis. The hypoxia-inducible factor–1 (HIF-1) transcriptionally up-regulates erythropoietin, transferrin, and transferrin receptor, leading to increased erythropoiesis and hematopoietic iron supply. To test the hypothesis that ALAS2 expression might be regulated by a similar mechanism, we exposed murine erythroleukemia cells to hypoxia (1% O2) and found an up to 3-fold up-regulation of ALAS2 mRNA levels and an increase in cellular heme content. A fragment of the ALAS2 promoter ranging from −716 to +1 conveyed hypoxia responsiveness to a heterologous luciferase reporter gene construct in transiently transfected HeLa cells. In contrast, iron depletion, known to induce HIF-1 activity but inhibit ALAS2 translation, did not increase ALAS2 promoter activity. Mutation of a previously predicted HIF-1–binding site (−323/−318) within this promoter fragment and DNA-binding assays revealed that hypoxic up-regulation is independent of this putative HIF-1 DNA-binding site.

scholarly journals Inhibitor of DNA Binding 1 Is Induced during Kidney Ischemia-Reperfusion and Is Critical for the Induction of Hypoxia-Inducible Factor-1α

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Dan Wen ◽  
Yan-Fang Zou ◽  
Yao-Hui Gao ◽  
Qian Zhao ◽  
Yin-Yin Xie ◽  
...  
Keyword(s):  
Dna Binding ◽  
Ischemia Reperfusion ◽  
Hypoxia Inducible Factor ◽  
Kidney Injury ◽  
Mrna Levels ◽  
Hypoxia Inducible Factor 1 ◽  
Renal Tubular ◽  
Inhibitor Of Dna Binding

In this study, rat models of acute kidney injury (AKI) induced by renal ischemia-reperfusion (I/R) and HK-2 cell models of hypoxia-reoxygenation (H/R) were established to investigate the expression of inhibitor of DNA binding 1 (ID1) in AKI, and the regulation relationship between ID1 and hypoxia-inducible factor 1 alpha (HIF-1α). Through western blot, quantitative real-time PCR, immunohistochemistry, and other experiment methods, the induction of ID1 after renal I/R in vivo was observed, which was expressed mainly in renal tubular epithelial cells (TECs). ID1 expression was upregulated in in vitro H/R models at both the protein and mRNA levels. Via RNAi, it was found that ID1 induction was inhibited with silencing of HIF-1α. Moreover, the suppression of ID1 mRNA expression could lead to decreased expression and transcription of HIF-1αduring hypoxia and reoxygenation. In addition, it was demonstrated that both ID1 and HIF-1αcan regulate the transcription of twist. This study demonstrated that ID1 is induced in renal TECs during I/R and can regulate the transcription and expression of HIF-1α.

lncRNA KCNQ1OT1 promotes the proliferation, migration and invasion of retinoblastoma cells by upregulating HIF-1α via sponging miR-153-3p

2020 ◽  
Vol 68 (8) ◽  
pp. 1349-1356
Author(s):  
Yujin Wang ◽  
Jixiang Wang ◽  
Hongyan Hao ◽  
Xiangxia Luo
Keyword(s):  
Colony Formation ◽  
Rna Binding ◽  
Hypoxia Inducible Factor ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Tissue Samples ◽  
Qrt Pcr ◽  
Gene Assay

It is reported that lncRNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) is oncogenic in many cancers. This work aimed at probing into its expression and biological functions in retinoblastoma (RB) as well as its regulatory effects on miR-153-3p and hypoxia-inducible factor-1α (HIF-1α). In our study, RB samples in pair were collected, and quantitative real-time PCR (qRT-PCR) was employed for examining the expression levels of KCNQ1OT1, miR-153-3p and HIF-1α. KCNQ1OT1 short hairpin RNAs were transfected into SO-Rb50 and HXO-RB44 cell to inhibit the expression of KCNQ1OT1. The proliferative activity, colony formation ability and apoptosis were examined through cell counting kit-8 assay, colony formation assays, Transwell assay and flow cytometry, respectively. qRT-PCR and western blot analysis were used for analyzing the changes of miR-153-3p and HIF-1α induced by KCNQ1OT1. The regulatory relationships between miR-153-3p and KCNQ1OT1, miR-153-3p and HIF-1α were examined by dual luciferase reporter gene assay and RNA-binding protein immunoprecipitation assay. The results of our study showed that KCNQ1OT1 expression was markedly enhanced in RB tissue samples, and KCNQ1OT1 knockdown had an inhibitory effect on the proliferation, migration, invasion and viability of RB cells. There were two validated binding sties between KCNQ1OT1 and miR-153-3p, and KCNQ1OT1 negatively regulated the expression of miR-153-3p in RB cells. HIF-1α was a target gene of miR-153-3p, and could be positively regulated by KCNQ1OT1. In conclusion, our study indicates that KCNQ1OT1 can increase the malignancy of RB cells via regulating miR-153-3p/HIF-1α axis.

scholarly journals Cyclin G2 Regulates Adipogenesis through PPARγ Coactivation

Endocrinology ◽  
2010 ◽  
Vol 151 (11) ◽  
pp. 5247-5254 ◽  
Author(s):  
Victor Aguilar ◽  
Jean-Sébastien Annicotte ◽  
Xavier Escote ◽  
Joan Vendrell ◽  
Dominique Langin ◽  
...  
Keyword(s):  
Adipocyte Differentiation ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Cell Cycle Regulators ◽  
Luciferase Reporter Gene ◽  
Peroxisome Proliferator Activated Receptor ◽  
Cyclin G2 ◽  
Regulatory Effects

Cell cycle regulators such as cyclins, cyclin-dependent kinases, or retinoblastoma protein play important roles in the differentiation of adipocytes. In the present paper, we investigated the role of cyclin G2 as a positive regulator of adipogenesis. Cyclin G2 is an unconventional cyclin which expression is up-regulated during growth inhibition or apoptosis. Using the 3T3-F442A cell line, we observed an up-regulation of cyclin G2 expression at protein and mRNA levels throughout the process of cell differentiation, with a further induction of adipogenesis when the protein is transiently overexpressed. We show here that the positive regulatory effects of cyclin G2 in adipocyte differentiation are mediated by direct binding of cyclin G2 to peroxisome proliferator-activated receptor γ (PPARγ), the key regulator of adipocyte differentiation. The role of cyclin G2 as a novel PPARγ coactivator was further demonstrated by chromatin immunoprecipitation assays, which showed that the protein is present in the PPARγ-responsive element of the promoter of aP2, which is a PPARγ target gene. Luciferase reporter gene assays, showed that cyclin G2 positively regulates the transcriptional activity of PPARγ. The role of cyclin G2 in adipogenesis is further underscored by its increased expression in mice fed a high-fat diet. Taken together, our results demonstrate a novel role for cyclin G2 in the regulation of adipogenesis.

scholarly journals cDNA cloning and expression of rat homeobox gene, Hex, and functional characterization of the protein

1999 ◽  
Vol 339 (1) ◽  
pp. 111-117 ◽  
Author(s):  
Takashi TANAKA ◽  
Tetsuya INAZU ◽  
Kazuya YAMADA ◽  
Zaw MYINT ◽  
Vincent W. KENG ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Rat Liver ◽  
Functional Characterization ◽  
Homeobox Gene ◽  
Hepatoma Cells ◽  
Cloning And Expression ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Cdna Clones

We isolated two cDNA clones of rat Hex, a homeobox protein, studied its expression in rat liver and various cells, and characterized the protein. The levels of Hex mRNA were only slightly increased in liver of rats refed with a high-carbohydrate diet or after partial hepatectomy. Whereas the expression of Hex mRNA was detected in hepatocytes isolated from adult rat liver and also in highly differentiated hepatoma cells, no Hex mRNA was detected in poorly differentiated hepatoma cells. Hex mRNA was also detected in liver from embryo aged 15 days. Expression of Hex was increased in F9 cells during differentiation into visceral endoderm cells by treatment with retinoic acid. This stimulation occurred prior to an increase in the level of α-fetoprotein mRNA. When fusion-protein expression vectors of GAL4 DNA-binding domain and Hex were co-transfected with luciferase reporter plasmid, with or without five copies of the GAL4-binding site, into HepG2 cells, the luciferase activities were decreased in concentration- and GAL4-binding site-dependent manners. This repression did not require the presence of the homeodomain, which is located between the amino acid residues 137 and 196. Its repression domain was mapped between the residues 45 and 136 in the proline-rich N-terminal region. In addition, the homeodomain was responsible for DNA-binding of Hex. These results indicate that Hex functions as a transcriptional repressor and may be involved in the differentiation and/or maintenance of the differentiated state in hepatocytes.

scholarly journals Downregulation of PU.1 Leads to Decreased Expression of Dectin-1 in Alveolar Macrophages during Pneumocystis Pneumonia

2010 ◽  
Vol 78 (3) ◽  
pp. 1058-1065 ◽  
Author(s):  
Chen Zhang ◽  
Shao-Hung Wang ◽  
Chung-Ping Liao ◽  
Shoujin Shao ◽  
Mark E. Lasbury ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Mrna Level ◽  
Gene Promoter ◽  
Pneumocystis Pneumonia ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Mobility Shift Assay

ABSTRACT Dectin-1 is an important macrophage phagocytic receptor recognizing fungal β-glucans. In this study, the mRNA levels of the Dectin-1 gene were found to be decreased by 61% in alveolar macrophages (AMs) from Pneumocystis-infected mice. The expression of Dectin-1 protein on the surface of these cells was also significantly decreased. By fluorescence in situ hybridization, mRNA expression levels of the transcription factor PU.1 were also found to be significantly reduced in AMs from Pneumocystis-infected mice. Electrophoretic mobility shift assay showed that PU.1 protein bound Dectin-1 gene promoter. With a luciferase reporter gene driven by the Dectin-1 gene promoter, the expression of the PU.1 gene in NIH 3T3 cells was found to enhance the luciferase activity in a dose-dependent manner. PU.1 expression knockdown by small interfering RNA (siRNA) caused a 63% decrease in Dectin-1 mRNA level and 40% decrease in protein level in AMs. Results of this study indicate that downregulation of PU.1 during Pneumocystis pneumonia leads to decreased expression of Dectin-1 in AMs.

Tumor necrosis factor-α inhibits peroxisome proliferator-activated receptor γ activity at a posttranslational level in hepatic stellate cells

2004 ◽  
Vol 286 (5) ◽  
pp. G722-G729 ◽  
Author(s):  
Chin K. Sung ◽  
Hongyun She ◽  
Shigang Xiong ◽  
Hidekazu Tsukamoto
Keyword(s):  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Luciferase Reporter ◽  
Critical Event ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Peroxisome Proliferator Activated Receptor ◽  
Tnf Α

Diminished activity of peroxisome proliferator-activated receptor γ (PPARγ) is implicated in activation of hepatic stellate cells (HSC), a critical event in the development of liver fibrosis. In the present study, we investigated PPARγ regulation by TNF-α in an HSC line designated as BSC. In BSC, TNF-α decreased both basal and ligand (GW1929)-induced PPARγ mRNA levels without changing its protein expression. Nuclear extracts from BSC treated with TNF-α showed decreased binding of PPARγ to PPAR-responsive element (PPRE) as determined by electrophoretic mobility shift assay. In BSC transiently transfected with a PPARγ1 expression vector and a PPRE-luciferase reporter gene, TNF-α decreased both basal and GW1929-induced transactivation of the PPRE promoter. TNF-α increased activation of ERK1/2 and JNK, previously implicated in phosphorylation of Ser82 of PPARγ1 and resultant negative regulation of PPARγ transactivity. In fact, TNF-α failed to inhibit transactivity of a Ser82Ala PPARγ1 mutant in BSC. TNF-α-mediated inhibition of PPARγ transactivity was not blocked with a Ser32Ala/Ser36Ala mutant of inhibitory NF-κBα (IκBα). These results suggest that TNF-α inhibits PPARγ transactivity in cultured HSC, at least in part, by diminished PPARγ-PPRE (DNA) binding and ERK1/2-mediated phosphorylation of Ser82 of PPARγ1, but not via the NF-κB pathway.

scholarly journals Allelic variant at −79 (C>T) in CDKN1B (p27Kip1) confers an increased risk of thyroid cancer and alters mRNA levels

2010 ◽  
Vol 17 (2) ◽  
pp. 317-328 ◽  
Author(s):  
Iñigo Landa ◽  
Cristina Montero-Conde ◽  
Donatella Malanga ◽  
Silvia De Gisi ◽  
Guillermo Pita ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Cancer Susceptibility ◽  
Recessive Model ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Luciferase Reporter Gene ◽  
Reverse Transcription Pcr ◽  
First Case ◽  
Common Genetic Variants ◽  
Increased Risk

The aim of this study is to assess if common genetic variants located in the CDKN1B locus, coding for the cell cycle inhibitor p27Kip1, are involved in thyroid cancer susceptibility. Based on the literature and functional predictions, we selected three polymorphisms within the CDKN1B gene (rs2066827 (T326G, V109G), rs34330 (−79C>T) and rs36228499 (−838C>A)) to perform the first case–control study in thyroid cancer involving this locus. We had 649 Spanish patients with sporadic thyroid cancer and 385 healthy representative controls available. Luciferase reporter gene assays, real-time quantitative reverse transcription-PCR and immunoblot experiments were carried out to demonstrate the putative effect of the associated variant. The polymorphism rs34330 (−79C>T) was identified as a risk factor for developing the follicular variant of papillary thyroid carcinoma (FVPTC), fitting a recessive model (odds ratio=2.12; 95% confidence interval=1.09–4.15; P value=0.023). The risk allele (T) of this single nucleotide polymorphism led to a lower transcription rate in cells transfected with a luciferase reporter driven by the polymorphic p27Kip1 promoter (P value <0.001). This effect was observed in −79TT genotype control carriers, who showed a tendency towards lower CDKN1B mRNA levels in lymphocytes, as well as at the protein level. This is the first study that identifies CDKN1B as a low-penetrance gene in thyroid cancer, and specifically in FVPTC subtype. We propose a reduced CDKN1B gene transcription depending on the genotype of the −79C>T (rs34330) variant as a novel mechanism underlying p27Kip1 downregulation.

scholarly journals Identification of PARP-1, Histone H1 and SIRT-1 as New Regulators of Breast Cancer-Related Aromatase Promoter I.3/II

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 427
Author(s):  
Alexander Kaiser ◽  
Thomas Krüger ◽  
Gabriele Eiselt ◽  
Joachim Bechler ◽  
Olaf Kniemeyer ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Binding ◽  
Cell Model ◽  
Histone H1 ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Aromatase Expression ◽  
Knock Out ◽  
Estrogen Synthesis ◽  
Parp 1

Paracrine interactions between malignant estrogen receptor positive (ER+) breast cancer cells and breast adipose fibroblasts (BAFs) stimulate estrogen biosynthesis by aromatase in BAFs. In breast cancer, mainly the cAMP-responsive promoter I.3/II-region mediates excessive aromatase expression. A rare single nucleotide variant (SNV) in this promoter region, which caused 70% reduction in promoter activity, was utilized for the identification of novel regulators of aromatase expression. To this end, normal and mutant promoter activities were measured in luciferase reporter gene assays. DNA-binding proteins were captured by DNA-affinity and identified by mass spectrometry. The DNA binding of proteins was analyzed using electrophoretic mobility shift assays, immunoprecipitation-based in vitro binding assays and by chromatin immunoprecipitation in BAFs in vivo. Protein expression and parylation were analyzed by western blotting. Aromatase activities and RNA-expression were measured in BAFs. Functional consequences of poly (ADP-ribose) polymerase-1 (PARP-1) knock-out, rescue or overexpression, respectively, were analyzed in murine embryonic fibroblasts (MEFs) and the 3T3-L1 cell model. In summary, PARP-1 and histone H1 (H1) were identified as critical regulators of aromatase expression. PARP-1-binding to the SNV-region was crucial for aromatase promoter activation. PARP-1 parylated H1 and competed with H1 for DNA-binding, thereby inhibiting its gene silencing action. In MEFs (PARP-1 knock-out and wild-type) and BAFs, PARP-1-mediated induction of the aromatase promoter showed bi-phasic dose responses in overexpression and inhibitor experiments, respectively. The HDAC-inhibitors butyrate, panobinostat and selisistat enhanced promoter I.3/II-mediated gene expression dependent on PARP-1-activity. Forskolin stimulation of BAFs increased promoter I.3/II-occupancy by PARP-1, whereas SIRT-1 competed with PARP-1 for DNA binding but independently activated the promoter I.3/II. Consistently, the inhibition of both PARP-1 and SIRT-1 increased the NAD+/NADH-ratio in BAFs. This suggests that cellular NAD+/NADH ratios control the complex interactions of PARP-1, H1 and SIRT-1 and regulate the interplay of parylation and acetylation/de-acetylation events with low NAD+/NADH ratios (reverse Warburg effect), promoting PARP-1 activation and estrogen synthesis in BAFs. Therefore, PARP-1 inhibitors could be useful in the treatment of estrogen-dependent breast cancers.

Bioengineered microRNA-1291 effects on pancreatic cancer cell proliferation and xenograft tumor growth.

2018 ◽  
Vol 36 (4_suppl) ◽  
pp. 307-307
Author(s):  
Mei-Juan Tu ◽  
Zhijian Duan ◽  
Qianyu Zhang ◽  
Jing-Xin Qiu ◽  
Frank J Gonzalez ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Growth ◽  
Therapeutic Potential ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Luciferase Reporter Gene ◽  
Protein Levels ◽  
Gene Assay

307 Background: MicroRNAs (miR) have proved to be vital regulators in the control of tumor progression. Our recent studies have revealed miR-1291 is downregulated in patient pancreatic cancer (PC) specimens and re-introduction of miR-1291 suppresses tumorigenesis of PC cells. We have developed a novel ncRNA bioengineering technology to produce a miR-1291 prodrug. In this study, we aimed to assess the effectiveness of this miR-1291 prodrug as a monotherapy, as well as in combination with chemotherapy, for treatment of PC. Methods: Sensitivity of PC cells to miR-1291 prodrug alone, gemcitabine plus nab-paclitaxel (Gem-nP) alone, and their combination was evaluated by CellTiter-Glo assay. Mature miR-1291 and ARID3B mRNA levels were determined by quantitative real-time PCR (q-PCR) assay. A luciferase reporter gene assay was used to validate interaction between miR-1291 and ARID3B 3’UTR. Target protein expression was examined by Western blot and immunofluorescence analyses. PANC-1 and PC patient-derived xenograft (PDX) mouse models were established and used to assess anti-tumor effects of miR-1291 monotherapy and combination therapy with Gem-nP. Results: Cytotoxicity assays showed that miR-1291 prodrug enhanced the sensitivity of PANC-1 and AsPC-1 cells to Gem-nP. Luciferase assays confirmed ARID3B as a target for miR-1291 as predicted by computational analysis. qPCR analysis demonstrated that miR-1291 prodrug was readily processed to mature miR-1291 and subsequently upregulated ARID3B mRNA levels. miR-1291 prodrug also elevated the protein levels of ARID3B. Co-administration of miR-1291 prodrug and Gem-nP increased caspase-3/7 and γH2AX levels in PC cells, compared to miR-1291 or Gem-nP treatment alone. In addition, systemic administration of in vivo-jet PEI formulated miR-1291 prodrug suppressed tumor growth in both a PANC-1 xenograft model and three PDX models, and largely enhanced the efficacy of Gem-nP. All treatments were well tolerated in mice in vivo. Conclusions: Our bioengineered miR-1291 prodrug has therapeutic potential as a monotherapy but also can act as a sensitizing agent to chemotherapy. This novel treatment approach should be further explored for PC.

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