scholarly journals The disruption of circadian clockwork in differentiating cells from rat reproductive tissues as identified by in vitro real-time monitoring system

2007 ◽  
Vol 193 (3) ◽  
pp. 413-420 ◽  
Author(s):  
Pei-Jian He ◽  
Masami Hirata ◽  
Nobuhiko Yamauchi ◽  
Seiichi Hashimoto ◽  
Masa-aki Hattori
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Monitoring System ◽  
Cell Types ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Peripheral Tissues ◽  
Reproductive Tissues ◽  
Clock Gene Expression

The circadian clock, regulating hormonal secretion and metabolisms in accordance with the environmental light–dark cycle, resides in almost all peripheral tissues as well as in the superchiasmatic nucleus. Clock gene expression has been found to be noncyclic during spermatogenesis and the differentiation of thymocytes. However, currently little is known about how cell differentiation could affect circadian clockwork. We performed this study using the in vitro real-time oscillation monitoring system to examine the clockwork in several types of differentiating cells originated from reproductive tissues of transgenic rats (constructed with Period gene 2 (Per2) promoter-destabilized luciferase reporter gene). After treatment with dexamethasone (DXM), persistent oscillation of Per2 expression was observed in both gonadotropin-induced and pregnant ovarian luteal cells, proliferative uterine stromal cells (USCs), and nondifferentiating testicular interstitial cells, with a cyclic period of ~24 h. In contrast to these cell types, only one cycle of oscillation was sustained in granulosa cells undergoing differentiation. Additionally, Per2 oscillation was irregular in USCs undergoing decidualization induced by medroxyprogesterone acetate plus N6, 2-O-dibutyryl adenosine 3′:5′-cyclic monophosphate. Furthermore, no oscillation of Per2 expression was evoked by DXM in Leydig cells and thymocytes. In conclusion, the present study characterized the oscillation of Per2 gene expression in several types of ovarian, uterine, and testicular cells, and it is strongly suggested that circadian clockwork is affected during cellular differentiation.

Development of a high throughput luciferase reporter gene system for screening activators and repressors of human collagen Iα2 gene expression

2015 ◽  
Vol 93 (10) ◽  
pp. 887-892 ◽  
Author(s):  
Rushita A. Bagchi ◽  
Viktoriya Mozolevska ◽  
Bernard Abrenica ◽  
Michael P. Czubryt
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Negative Impact ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Reporter System ◽  
Lead Compounds ◽  
Col1a2 Gene ◽  
Human Collagen

Fibrosis, which is characterized by the excessive production of matrix proteins, occurs in multiple tissues and is associated with increased morbidity and mortality. Despite its significant negative impact on patient outcomes, therapies targeted to treat fibrosis are currently lacking. Screening for inhibitors of the expression of collagen, the primary component of fibrotic lesions, represents an option for the identification of novel lead compounds for therapeutic development with potentially fewer off-target effects compared with the targeting of multifunctional cell signaling pathways. Here we report on the generation of a stable luciferase reporter system using a fibroblast cell line, which can be used for rapidly screening both activators and repressors of human collagen COL1A2 gene transcription in a high throughput setting. This in vitro screening tool was validated using known agonists (scleraxis, TGF-β, angiotensin II, CTGF) and antagonists (TNF-α, pirfenidone) of COL1A2 gene expression. The COL1A2-luc NIH-3T3 fibroblast system provides a useful and effective screen for potential lead compounds with pro- or anti-fibrotic properties.

scholarly journals Novel Luciferase Reporter System for In Vitro and Organ-Specific Monitoring of Differential Gene Expression in Listeria monocytogenes

2006 ◽  
Vol 72 (4) ◽  
pp. 2876-2884 ◽  
Author(s):  
Peter A. Bron ◽  
Ian R. Monk ◽  
Sinéad C. Corr ◽  
Colin Hill ◽  
Cormac G. M. Gahan
Keyword(s):  
Gene Expression ◽  
Listeria Monocytogenes ◽  
Real Time ◽  
Single Copy ◽  
Luciferase Reporter ◽  
Exponential Growth Phase ◽  
Fatty Aldehyde ◽  
Reporter System ◽  
Study Gene Expression

ABSTRACT In this paper we describe construction of a luciferase-based vector, pPL2lux, and use of this vector to study gene expression in Listeria monocytogenes. pPL2lux is a derivative of the listerial integration vector pPL2 and harbors a synthetic luxABCDE operon encoding a fatty acid reductase complex (LuxCDE) involved in synthesis of the fatty aldehyde substrate for the bioluminescence reaction catalyzed by the LuxAB luciferase. We constructed pPL2lux derivatives in which the secA and hlyA promoters were translationally fused to luxABCDE and integrated as a single copy into the chromosome of L. monocytogenes EGD-e. Growth experiments revealed that hlyA was expressed predominantly in the stationary phase in LB medium buffered at pH 7.4, whereas secA expression could be detected in the exponential growth phase. Moreover, the correlation between luciferase activity and transcription levels, as determined by reverse transcriptase PCR, was confirmed using conditions known to lead to repression and activation of hemolysin expression (addition of cellobiose and activated charcoal, respectively). Furthermore, hemolysin expression could be monitored in real time during invasion of an intact monolayer of C2Bbe1 (Caco-2-derived) cells. Finally, hemolysin expression could be detected in the livers, spleens, and kidneys of mice 3 days postinfection. These experiments clearly established the effectiveness of pPL2lux as a quantitative reporter system for real-time, noninvasive evaluation of gene expression in L. monocytogenes.

scholarly journals Real-time monitoring of gene expression by luciferase reporter gene.

1996 ◽  
Vol 36 (6) ◽  
pp. 289-292
Author(s):  
Takao KONDO ◽  
Noboru YAMAGUCHI ◽  
Masahiro ISHIURA
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Real Time Monitoring ◽  
Luciferase Reporter Gene

scholarly journals SUN-267 Dissecting Type 2 CRH Receptor Signaling Characteristics in the Hypothalamic Cell Line MHYPOA-2/30

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Viridiana Alcantara-Alonso ◽  
Patricia de Gortari ◽  
Robert Dallmann ◽  
Dimitris Grammatopoulos
Keyword(s):  
Gene Expression ◽  
Plasma Membrane ◽  
Protein Expression ◽  
Cell Line ◽  
Cellular Localization ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Luciferase Reporter Gene

Abstract The stress peptides coticotropin-releasing hormone (CRH) and urocortins (Ucns) exert anorectic effects acting mainly through the type 2 CRH receptor (CRH-R2) in the hypothalamus. Impairment of CRH-R2 signaling in chronically stressed rats has been linked with the development of hyperphagia (Alcantara-Alonso et al. Neuropeptides, 2017) however the exact mechanisms and molecular defects are unknown. In the present study we used the mHypoA-2/30, a hypothalamic immortalized cell line derived from adult mice (Belsham et al. FASEB J, 2009) to further explore the signaling molecules mediating the anorexigenic effect of the CRH-R2 cognate agonist urocortin 2 (Ucn2). Specifically, we investigated mRNA, protein expression and cellular localization of CRH-R2 in the mHypoA-2/30 neurons. Additionally, we examined the effects of Ucn2 on the phosphorylation of CREB and AMPK, as well as its transcriptional effects on genes of feeding-related peptides and molecules involved in modulation of circadian rhythms. Both CRH-R2 mRNA and protein expression were detected in mHypoA-2/30; indirect immunoflourescence experiments using a specific CRH-R2 antibody demonstrated widespread localization in the plasma membrane and cytoplasm. Moreover, the receptor sub-cellular localization was redistributed in response to activation by Ucn2 (100 nM), as the plasma membrane immunofluorescent signal was decreased after 4h of agonist treatment, suggesting CRH-R2 homologous internalization. We also observed a 50% increase in the phosphorylation of CREB associated with a concomitant decrease in AMPK phosphorylation after 30 min of Ucn2 treatment. Among the panel of hypothalamic genes analyzed, we identified after 24h of Ucn2 treatment increases in the gene expression of the anorexigenic peptides neurotensin and proopiomelanocortin. Interestingly, sustained CRH-R2 activation also led to an increase in the mRNA levels of Aryl Hydrocarbon Receptor Nuclear Translocator Like (ARNTL), a protein involved in the control of circadian rhythm. A luciferase reporter gene analysis of ARNTL showed that the mHypoA-2/30 cells also exhibit circadian patterns of expression and that the treatment with Ucn2 enhanced circadian amplitude of ARNTL reporter on these cells, which in turn may be involved in glucocorticoid release in circadian cycles and stimulating appetite during the activity phase of the animals. In conclusion, we found that the mHypoA-2/30 cell line expresses endogenous functional CRH-R2 that is linked to downstream regulation of anorexigenic gene expression. This cell line appears to be a useful in vitro tool to study hypothalamic CRH-R2 signaling machinery involved in central control of food intake and circadian cycles.

scholarly journals Differential regulation of the TRH gene promoter by triiodothyronine and dexamethasone in pancreatic islets

2001 ◽  
Vol 170 (1) ◽  
pp. 91-98 ◽  
Author(s):  
P Fragner ◽  
SL Lee ◽  
S Aratan de Leon
Keyword(s):  
Gene Expression ◽  
Pancreatic Islets ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Flanking Sequence ◽  
Luciferase Reporter Gene ◽  
Neoplastic Cells

TRH was initially found in the hypothalamus and regulates TSH secretion. TRH is also produced by insulin-containing beta-cells. Endogenous TRH positively regulates glucagon secretion and attenuates pancreatic exocrine secretion. We have previously shown that triiodothyronine (T(3)) down-regulates pre-pro-TRH gene expression in vivo and in vitro. The present study was designed to determine the initial impact of T(3) on rat TRH gene promoter and to compare this effect with that of dexamethasone (Dex). Primary islet cells and neoplastic cells (HIT T-15 and RIN m5F) were transiently transfected with fragments of the 5'-flanking sequence of TRH fused to the luciferase reporter gene. The persistence of high TRH concentrations in fetal islets in culture, probably due to transactivating factors, allowed us to explore how T(3) and Dex regulate the TRH promoter activity in transfected cells and whether the hormone effect is dependent on the cell type considered. TRH gene promoter activity is inhibited by T(3) in primary but not neoplastic cells and stimulated by Dex in both primary and neoplastic cells of islets. These findings validate previous in vivo and in vitro studies and indicate the transcriptional impact of these hormones on TRH gene expression in the pancreatic islets.

scholarly journals Extrachromosomal circular DNA, microDNA, without canonical promoters produce short regulatory RNAs that suppress gene expression

2019 ◽  
Author(s):  
Teressa Paulsen ◽  
Yoshiyuki Shibata ◽  
Pankaj Kumar ◽  
Laura Dillon ◽  
Anindya Dutta
Keyword(s):  
Gene Expression ◽  
Promoter Sequence ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Circular Dna ◽  
Endogenous Gene ◽  
Mrna Targets ◽  
Small Regulatory Rna

ABSTRACTInterest in extrachromosomal circular DNA (eccDNA) molecules has increased recently because of their widespread presence in normal cells across every species ranging from yeast to humans, their increased levels in cancer cells, and their overlap with oncogenic and drug-resistant genes. However, the majority of eccDNA (microDNA) are too small to carry protein coding genes. We have tested functional capabilities of microDNA, by creating artificial microDNA molecules mimicking known microDNA sequences and have discovered that they express functional small regulatory RNA including microRNA and novel si-like RNA. MicroDNA is transcribed in vitro and in vivo independent of a canonical promoter sequence. MicroDNA which carry miRNA genes form transcripts which are processed into mature miRNA molecules, through the endogenous RNA-interference pathway, which repress a luciferase reporter gene as well as endogenous mRNA targets of the miRNA. Further, microDNA containing sequences of exons repress the endogenous gene from which the microDNA was derived through the formation of novel si-like RNA. We also show that endogenous microDNA associate with RNA polymerases subunits POLR2H and POLR3F. Together, these results suggest that microDNA may modulate gene expression through the production of both known and novel regulatory small RNA.

Benzisothiazolone Derivatives Exhibit Cytotoxicity in Hodgkin’s Lymphoma Cells through NF-κB Inhibition and are Synergistic with Doxorubicin and Etoposide

2020 ◽  
Vol 20 (6) ◽  
pp. 715-723
Author(s):  
Natarajan Nandakumar ◽  
Pushparathinam Gopinath ◽  
Jacob Gopas ◽  
Kannoth M. Muraleedharan
Keyword(s):  
Synergistic Effect ◽  
Hodgkin’S Lymphoma ◽  
A549 Cells ◽  
Hodgkin's Lymphoma ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Lymphoma Cells ◽  
Nuclear Extracts ◽  
Gene Assay

Background: The authors investigated the NF-κB inhibitory role of three Benzisothiazolone (BIT) derivatives (1, 2 and 3) in Hodgkin’s Lymphoma cells (L428) which constitutively express activated NF-κB. All three compounds showed dose-dependent NF-κB inhibition (78.3, 70.7 and 34.6%) in the luciferase reporter gene assay and were found cytotoxic at IC50 values of 3.3μg/ml, 4.35μg/ml and 13.8μg/ml, respectively by the XTT assay. BIT 1and BIT 2 (but not BIT 3) suppressed both NF-κB subunits p50 and p65 in cytoplasmic and nuclear extracts in a concentration-dependent manner. Furthermore, BIT 1 showed a moderate synergistic effect with the standard chemotherapy drugs etoposide and doxorubicin, whereas BIT 2 and 3 showed a moderate additive effect to antagonistic effect. Cisplatin exhibited an antagonist effect on all the compounds tested under various concentrations, except in the case of 1.56μg/ml of BIT 3 with 0.156μg/ml of cisplatin. The compounds also inhibited the migration of adherent human lung adenocarcinoma cells (A549) in vitro. We conclude that especially BIT 1 and BIT 2 have in vitro anti-inflammatory and anti-cancer activities, which can be further investigated for future potential therapeutic use. Methods: Inspired by the electrophilic sulfur in Nuphar alkaloids, monomeric and dimeric benzisothiazolones were synthesized from dithiodibenzoic acid and their NF-κB inhibitory role was explored. NF-κB inhibition and cytotoxicity of the synthesized derivatives were studied using luciferase reporter gene assay and XTTassay. Immunocytochemistry studies were performed using L428 cells. Cell migration assay was conducted using the A549 cell line. L428 cells were used to conduct combination studies and the results were plotted using CompuSyn software. Results: Benzisothiazolone derivatives exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. Potent compounds showed suppression of both NF-κB subunits p50 and p65 in a concentrationdependent manner, both in cytoplasmic and nuclear extracts. Combination studies suggest that benzisothiazolone derivatives possess a synergistic effect with etoposide and doxorubicin. Furthermore, the compounds also inhibited the migration of A549 cells. Conclusion: Benzisothiazolones bearing one or two electrophilic sulfur atoms as part of the heterocyclic framework exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. In addition, these derivatives also exhibited a synergistic effect with etoposide and doxorubicin along with the ability to inhibit the migration of A549 cells. Our study suggests that BIT-based new chemical entities could lead to potential anticancer agents.

scholarly journals Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 11-26
Author(s):  
Maike Busch ◽  
Natalia Miroschnikov ◽  
Jaroslaw Thomas Dankert ◽  
Marc Wiesehöfer ◽  
Klaus Metz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treated Patient ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

scholarly journals MiR-206 regulates the progression of osteoporosis via targeting HDAC4

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Zhiyuan Lu ◽  
Dawei Wang ◽  
Xuming Wang ◽  
Jilong Zou ◽  
Jiabing Sun ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Diagnostic Value ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Mineral Density ◽  
Gene Assay

Abstract Background More and more studies have confirmed that miRNAs play an important role in maintaining bone remodeling and bone metabolism. This study investigated the expression level of miR-206 in the serum of osteoporosis (OP) patients and explored the effect and mechanism of miR-206 on the occurrence and development of osteoporosis. Methods 120 postmenopausal women were recruited, including 63 cases with OP and 57 women without OP. The levels of miR-206 were determined by qRT-PCR technology. Spearman correlation coefficient was used to evaluate the correlation of miR-206 with bone mineral density (BMD). An ROC curve was used to evaluate the diagnostic value of miR-206 in osteoporosis. The effects of miR-206 on cell proliferation and cell apoptosis of hFOBs were measured by CCK-8 assay and flow cytometry, respectively. Luciferase reporter gene assay was used to confirm the interaction of miR-206 and the 3′UTR of HDAC4. Results Serum miR-206 had low expression level in osteoporosis patient group compared with control group. The expression level of serum miR-206 had diagnostic value for osteoporosis, and the serum miR-206 levels were positively correlated with BMD. The down-regulated miR-206 could inhibit cell proliferation and promote cell apoptosis. Luciferase analysis indicated that HDAC4 was the target gene of miR-206. Conclusions MiR-206 could be used as a new potential diagnostic biomarker for osteoporosis, and in in vitro cell experiments, miR-206 may regulate osteoblast cell proliferation and apoptosis by targeting HDAC4.

scholarly journals Mechanism of MicroRNA-375 Promoter Methylation in Promoting Ovarian Cancer Cell Malignancy

2021 ◽  
Vol 20 ◽  
pp. 153303382098011
Author(s):  
Junjun Shu ◽  
Ling Xiao ◽  
Sanhua Yan ◽  
Boqun Fan ◽  
Xia Zou ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Protein Expression ◽  
Cell Lines ◽  
Cell Invasion ◽  
Promoter Methylation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Cell Malignancy

Objective: Ovarian cancer (OC) ranks one of the most prevalent fatal tumors of female genital organs. Aberrant promoter methylation triggers changes of microRNA (miR)-375 in OC. Our study aimed to evaluate the mechanism of methylated miR-375 promoter region in OC cell malignancy and to seek the possible treatment for OC. Methods: miR-375 promoter methylation level in OC tissues and cells was detected. miR-375 expression in OC tissues and cell lines was compared with that in demethylated cells. Role of miR-375 in OC progression was measured. Dual-luciferase reporter gene assay was utilized to verify the targeting relationship between miR-375 and Yes-associated protein 1 (YAP1). Then, Wnt/β-catenin pathway-related protein expression was tested. Moreover, xenograft transplantation was applied to confirm the in vitro experiments. Results: Highly methylated miR-375 was seen in OC tissues and cell lines, while its expression was decreased as the promoter methylation increased. Demethylation in OC cells brought miR-375 back to normal level, with obviously declined cell invasion, migration and viability and improved apoptosis. Additionally, miR-375 targeted YAP1 to regulate the Wnt/β-catenin pathway protein expression. Overexpressed YAP1 reversed the protein expression, promoted cell invasion, migration and viability while reduced cell apoptosis. Overexpressed miR-375 in vivo inhibited OC progression. Conclusion: Our study demonstrated that demethylated miR-375 inhibited OC growth by targeting YAP1 and downregulating the Wnt/β-catenin pathway. This investigation may offer novel insight for OC treatment.

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