scholarly journals Optical Imaging of Tumor Hypoxia and Evaluation of Efficacy of a Hypoxia-Targeting Drug in Living Animals

2005 ◽  
Vol 4 (3) ◽  
pp. 153535002005051 ◽  
Author(s):  
Hiroshi Harada ◽  
Shinae Kizaka-Kondoh ◽  
Masahiro Hiraoka
Keyword(s):  
Cancer Therapy ◽  
Optical Imaging ◽  
Solid Tumors ◽  
Imaging System ◽  
Tumor Hypoxia ◽  
Immunohistochemical Analysis ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Construct

Solid tumors containing more hypoxic regions show a more malignant phenotype by increasing the expression of genes encoding angiogenic and metastatic factors. Hypoxia-inducible factor-1 (HIF-1) is a master transcriptional activator of such genes, and thus, imaging and targeting hypoxic tumor cells where HIF-1 is active are important in cancer therapy. In the present study, HIF-1 activity was monitored via an optical in vivo imaging system by using a luciferase reporter gene under the regulation of an artificial HIF-1-dependent promoter, 5HRE. To monitor tumor hypoxia, we isolated a stable reporter-transfectant, HeLa/5HRE-Luc, which expressed more than 100-fold luciferase in response to hypoxic stress, and observed bioluminescence from its xenografts. Immunohistochemical analysis of the xenografts with a hypoxia marker, pimonidazole, confirmed that the luciferase-expressing cells were hypoxic. Evaluation of the efficacy of a hypoxia-targeting prodrug, TOP3, using this optical imaging system revealed that hypoxic cells were significantly diminished by TOP3 treatment. Immunohistochemical analysis of the TOP3-treated xenografts confirmed that hypoxic cells underwent apoptosis and were removed after TOP3 treatment. These results demonstrate that this model system using the 5HRE-luciferase reporter construct provides qualitative information (hypoxic status) of solid tumors and enables one to conveniently evaluate the efficacy of cancer therapy on hypoxia in malignant solid tumors.

scholarly journals Tissue- and time-dependent estrogen receptor activation in estrogen reporter mice

2004 ◽  
Vol 32 (3) ◽  
pp. 689-701 ◽  
Author(s):  
JG Lemmen ◽  
RJ Arends ◽  
AL van Boxtel ◽  
PT van der Saag ◽  
B van der Burg
Keyword(s):  
Reporter Gene ◽  
Imaging System ◽  
Luciferase Activity ◽  
Estrogenic Activity ◽  
Receptor Activation ◽  
Time Dependent ◽  
Luciferase Reporter ◽  
In Vivo Model ◽  
Luciferase Reporter Gene

With the aim of developing an in vivo model that directly detects activation of estrogen receptors (ERs), transgenic mice carrying a luciferase reporter gene were generated. The luciferase reporter gene was under the control of three consensus estrogen-responsive elements (EREs) coupled to a minimal TATA-box, with or without flanking chick beta-globin insulators. By using this model in combination with the IVIS imaging system, in vivo ER activation was measured. Dose- and time-dependent luciferase activity was induced in various organs of adult transgenic male mice exposed to diethylstilbestrol (DES) (10-1000 micro g/kg) and 17beta-estradiol dipropionate (EP) (10-1000 micro g/kg), when luciferase activity was measured ex vivo. The highest (>10 000-fold) induction of luciferase was measured in bone and kidney 24 h after exposure to 1000 micro g/kg EP. Other highly responsive organs include liver, testis, pituitary, brain, prostate and colon, which show different activity profiles. This in vivo model for detecting estrogenic activity can be used to assess tissue-specific action of ER agonists and antagonists. These could include selective ER modulators and environmental estrogens. In combination with the IVIS imaging system, this in vivo model is a powerful tool for assessing the kinetics of gene activation by estrogenic compounds.

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.

In vitro and in vivo analysis of murine lipoprotein lipase gene promoter: tissue-specific expression

1995 ◽  
Vol 268 (2) ◽  
pp. E213-E218 ◽  
Author(s):  
J. M. Gimble ◽  
X. Hua ◽  
F. Wanker ◽  
C. Morgan ◽  
C. Robinson ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Lipoprotein Lipase ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Brown Adipose ◽  
Octamer Motif

Lipoprotein lipase, an enzyme of central importance to lipid metabolism, is most abundant in adipose tissues, cardiac and skeletal muscle, and portions of the brain. The current work examined the murine lipoprotein lipase promoter using transient transfection, gel-retention analyses, and transgenic mice. Maximum expression of the luciferase reporter gene in transfected cells was observed with -101 bp of the promoter. Nuclear extracts from tissues expressing lipoprotein lipase contained DNA binding proteins that recognize the CCAAT box (-64 bp) and an octamer motif (-46 bp); this combination of factors was absent in nonexpressing tissues. Transgenic mice from three of five founders prepared with -1,824-bp promoter constructs expressed the luciferase reporter gene at highest levels in brown adipose tissue and brain. These findings suggest that the -1,824-bp promoter region contains sequence elements responsible for the tissue-specific transcription of lipoprotein lipase in vivo.

scholarly journals LINC00839 Knockdown Restrains The Metastatic Behaviors of Nasopharyngeal Carcinoma By Sponging miR-454-3p

2021 ◽  
Author(s):  
Feng Ying Zhang ◽  
Xia Li ◽  
Ting Ting Huang ◽  
Mei Ling Xiang ◽  
Lin Lin Sun ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Non Coding Rna ◽  
Invasive Capacity

Abstract Background Long intergenic non-coding RNA 00839 (LINC00839) has been verified as a cancer-promoting gene in malignancies. However, the significance of LINC00839 in nasopharyngeal carcinoma (NPC) has yet to be elaborated, as well as its underlying mechanism.Methods LINC00839 and miR-454-3p relative expression levels in NPC cells were examined by qRT-PCR. The growth of cells was examined by CCK-8 and colony formation assays. Cell migration and invasion were examined by wound healing and Transwell experiment, respectively. The binding sequence of LINC00839 and miR-454-3p was confirmed by the luciferase reporter gene experiment. The regulatory function of LINC00839 and miR-454-3p on c-Met was investigated by western blot.Results Here, we revealed that LINC00839 was elevated in NPC. Both LINC00839 knockdown and upregulation of miR-454-3p suppressed NPC cells proliferation, invasive capacity and EMT in vitro. Besides, LINC00839 was validated as a miR-454-3p “sponge”, and upregulation of LINC00839 could reverse miR-454-3p-mediated functions in NPC C666-1 and SUNE-1 cells. Furthermore, c-Met was determined to be targeted by miR-454-3p. Notably, c-Met was downregulated by LINC00839 knockdown through sponging miR-454-3p. In vivo, LINC00839 knockdown resulted in a slower tumor growth.Conclusions Altogether, knockdown of LINC00839 inhibits the aggressive properties of NPC cells via sponging miR-454-3p and regulating c-Met.

scholarly journals Circular RNA CDR1as Inhibits the Metastasis of Gastric Cancer through Targeting miR-876-5p/GNG7 Axis

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Jiajia Jiang ◽  
Rong Li ◽  
Junyi Wang ◽  
Jie Hou ◽  
Hui Qian ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Epithelial Mesenchymal Transition ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Mesenchymal Transition ◽  
Underlying Mechanisms

Circular RNA CDR1as has been demonstrated to participate in various cancer progressions as miRNA sponges. The exact underlying mechanisms of CDR1as on gastric cancer (GC) metastasis remain unknown. Here, we found that CDR1as knockdown facilitated GC cell migration and invasion while its overexpression inhibited the migration and invasion abilities of GC cells in vitro and in vivo. Moreover, epithelial-mesenchymal transition- (EMT-) associated proteins and MMP2 and MMP9 were downregulated by CDR1as. Bioinformatics analysis combined with dual-luciferase reporter gene assays, western blot, RT-qPCR analysis, and functional rescue experiments demonstrated that CDR1as served as a miR-876-5p sponge and upregulated the target gene GNG7 expression to suppress GC metastasis. In summary, our findings indicate that CDR1as suppresses GC metastasis through the CDR1as/miR-876-5p/GNG7 axis.

In vivo regulation of β-MHC gene in rodent heart: role of T3 and evidence for an upstream enhancer

1999 ◽  
Vol 276 (4) ◽  
pp. C883-C891 ◽  
Author(s):  
Carola E. Wright ◽  
F. Haddad ◽  
A. X. Qin ◽  
P. W. Bodell ◽  
K. M. Baldwin
Keyword(s):  
Gene Expression ◽  
Thyroid Hormone ◽  
Reporter Gene ◽  
Normal Control ◽  
Transcriptional Activity ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Promoter Construct ◽  
Hormone Responsiveness

Cardiac β-myosin heavy chain (β-MHC) gene expression is mainly regulated through transcriptional processes. Although these results are based primarily on in vitro cell culture models, relatively little information is available concerning the interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart. Using a direct gene transfer approach, we studied the in vivo transcriptional activity of different-length β-MHC promoter fragments in normal control and in altered thyroid states. The test β-MHC promoter was fused to a firefly luciferase reporter gene, whereas the control α-MHC promoter was fused to the Renilla luciferase reporter gene and was used to account for variations in transfection efficiency. Absolute reporter gene activities showed that β- and α-MHC genes were individually and reciprocally regulated by thyroid hormone. The β-to-α ratios of reporter gene expression demonstrated an almost threefold larger β-MHC gene expression in the longest than in the shorter promoter fragments in normal control animals, implying the existence of an upstream enhancer. A mutation in the putative thyroid response element of the −408-bp β-MHC promoter construct caused transcriptional activity to drop to null. When studied in the −3,500-bp β-MHC promoter, construct activity was reduced (∼100-fold) while thyroid hormone responsiveness was retained. These findings suggest that, even though the bulk of the thyroid hormone responsiveness of the gene is contained within the first 215 bp of the β-MHC promoter sequence, the exact mechanism of triiodothyronine (T3) action remains to be elucidated.

CircRNA_0092516 regulates chondrocyte proliferation and apoptosis in osteoarthritis through the miR-337-3p/PTEN axis

2020 ◽  
Author(s):  
Zhihui Huang ◽  
Wenming Ma ◽  
Jinhuai Xiao ◽  
Xiaoyu Dai ◽  
Weiqi Ling
Keyword(s):  
Cell Apoptosis ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Mechanism Analysis ◽  
Potential Mechanism ◽  
Luciferase Reporter Gene ◽  
Chondrocyte Proliferation ◽  
In Vivo Experiments

Abstract The dysregulation of circular RNAs (circRNAs) has been identified in various human diseases. Here, we probed into the potential mechanism of circRNA_0092516 in osteoarthritis (OA). The expression of circRNA_0092516 was tested by quantitative real-time PCR. MTT, flow cytometry and western blot were applied to confirm the functions of circRNA_0092516 in vitro. Besides, RNA pull-down and dual-luciferase reporter gene experiments were applied to probe into the mechanism. circRNA_0092516 was raised in the tissues of OA patients and chondrocytes stimulated by IL-1β. The potential mechanism analysis expounded that circRNA_0092516 bound to miR-337-3p, and the interference with circRNA_0092516 boosted chondrocyte proliferation and restrained cell apoptosis through the miR-337-3p/phosphatase and tensin homolog (PTEN) axis, thereby improving OA. In-vivo experiments expounded that circRNA_0092516 regulated cartilage production through miR-337-3p. Overall, our data expounded that the interference with circRNA_0092516 boosted chondrocyte proliferation and restrained cell apoptosis through the miR-337-3p/PTEN axis, eventually slowed down the progress of OA.

scholarly journals Multimodal optical imaging system for in vivo investigation of cerebral oxygen delivery and energy metabolism

2015 ◽  
Vol 6 (12) ◽  
pp. 4994 ◽  
Author(s):  
Mohammad A. Yaseen ◽  
Vivek J. Srinivasan ◽  
Iwona Gorczynska ◽  
James G. Fujimoto ◽  
David A. Boas ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Optical Imaging ◽  
Oxygen Delivery ◽  
Imaging System ◽  
Cerebral Oxygen ◽  
Optical Imaging System

scholarly journals Posttranscriptional mechanisms involving microRNA-27a and b contribute to fast-specific and glucocorticoid-mediated myostatin expression in skeletal muscle

2011 ◽  
Vol 300 (1) ◽  
pp. C124-C137 ◽  
Author(s):  
David L. Allen ◽  
Amanda S. Loh
Keyword(s):  
Receptor Expression ◽  
Luciferase Reporter ◽  
Potential Contribution ◽  
Recognition Sequence ◽  
Luciferase Reporter Construct ◽  
Glucocorticoid Treatment ◽  
Wide Range ◽  
Expression Construct

Expression of the antigrowth factor myostatin (MSTN) differs between fast and slow skeletal muscles and is increased in nearly every form of muscle atrophy, but the contribution of transcriptional vs. posttranscriptional mechanisms to its differing expression in these states has not been defined. We show here that levels of mature MSTN mRNA were sixfold greater in fast vs. slow muscle and were increased twofold in fast muscle in response to dexamethasone (Dex) injection in vivo and in C2C12 myotubes following Dex treatment in vitro, but that levels of MSTN pre-mRNA, a readout of transcription, only minimally and nonsignificantly differed in these states. Moreover, Dex treatment with or without cotransfection with a glucocorticoid receptor expression construct had only modest effects on mouse MSTN promoter activity in C2C12 myotubes. We therefore explored the potential contribution of posttranscriptional mechanisms, and the role of the microRNAs miR-27a and b in particular, on MSTN expression. The MSTN 3′-untranslated region (UTR) contains a putative recognition sequence for miR-27a and b that is conserved across a wide range of vertebrate species. Cotransfection of a MSTN 3′-UTR-luciferase construct with a miR-27b expression construct significantly attenuated by approximately half while mutation of the miR-27 recognition sequence significantly increased by approximately twofold the activity of a MSTN 3′-UTR construct and decreased mRNA degradation of a luciferase reporter construct in C2C12 myotubes. Expression of miR-27a and b was almost sixfold greater in slow-twitch than in fast-twitch muscle in vivo, and miR-27a expression was significantly decreased by nearly half by glucocorticoid treatment in vitro. Finally, the miR-27a and b promoters were activated by cotransfection with the slow-specific signaling molecules calcineurin and peroxisome proliferator-activated receptor-γ coactivator-1α. The present data represent the first demonstration that posttranscriptional mechanisms involving miR-27a and b may contribute to fast-specific and glucocorticoid-dependent myostatin expression in muscle.

scholarly journals Xiao Yao San against Corticosterone-Induced Stress Injury via Upregulating Glucocorticoid Receptor Reaction Element Transcriptional Activity

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Guoping Cao ◽  
Shenglan Gong ◽  
Fengxue Zhang ◽  
Wenjun Fu
Keyword(s):  
Hippocampal Neurons ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Luciferase Reporter ◽  
Potential Candidate ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Stress Injury ◽  
Induced Stress

Previous studies have revealed that uncontrollable stress can impair the synaptic plasticity and firing property of hippocampal neurons, which influenced various hippocampal-dependent tasks including memory, cognition, behavior, and mood. In this work, we had investigated the effects and mechanisms of the Chinese herbal medicine Xiao Yao San (XYS) against corticosterone-induced stress injury in primary hippocampal neurons (PHN) cells. We found that XYS and RU38486 could increase cell viabilities and decrease cell apoptosis by MTT, immunofluorescence, and flow cytometry assays. In addition, we observed that XYS notably inhibited the nuclear translocation of GR and upregulated the mRNA and protein expressions levels of Caveolin-1, GR, BDNF, TrkB, and FKBP4. However, XYS downregulated the FKBP51 expressions. Furthermore, the results of the electrophoretic mobility shift assay (EMSA) and double luciferase reporter gene detection indicated that FKBP4 promotes the transcriptional activity of GR reaction element (GRE) by binding with GR, and FKBP51 processed the opposite action. Thein vivoexperiment also proved the functions of XYS. These results suggested that XYS showed an efficient neuroprotection against corticosterone-induced stress injury in PHN cells by upregulating GRE transcriptional activity, which should be developed as a potential candidate for treating stress injury in the future.

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