Design, synthesis and biochemical investigation, by in vitro luciferase reporter system, of peptide nucleic acids as new inhibitors of miR-509-3p involved in the regulation of cystic fibrosis disease-gene expression

MedChemComm ◽  
2014 ◽  
Vol 5 (1) ◽  
pp. 68-71 ◽  
Author(s):  
Felice Amato ◽  
Rossella Tomaiuolo ◽  
Nicola Borbone ◽  
Ausilia Elce ◽  
Jussara Amato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Disease Gene ◽  
Peptide Nucleic Acids ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Design Synthesis ◽  
Biochemical Investigation ◽  
Luciferase Reporter System

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 Transcriptional slippage prompts recoding in alternate reading frames in the hepatitis C virus (HCV) core sequence from strain HCV-1

2008 ◽  
Vol 89 (7) ◽  
pp. 1569-1578 ◽  
Author(s):  
Maxime Ratinier ◽  
Steeve Boulant ◽  
Christophe Combet ◽  
Paul Targett-Adams ◽  
John McLauchlan ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Infectious Clone ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Subgenomic Replicon ◽  
Luciferase Reporter System ◽  
Transcriptional Slippage

Since the first report of frameshifting in HCV-1, its sequence has been the paradigm for examining the mechanism that directs alternative translation of the hepatitis C virus (HCV) genome. The region encoding the core protein from this strain contains a cluster of 10 adenines at codons 8–11, which is thought to direct programmed ribosomal frameshifting (PRF), but formal evidence for this process has not been established unequivocally. To identify the mechanisms of frameshifting, this study used a bicistronic dual luciferase reporter system in a coupled transcription/translation in vitro assay. This approach revealed +1 as well as –1 frameshifting, whereas point mutations, selectively introduced between codons 8 and 11, demonstrated that PRF did not readily account for frameshifting in strain HCV-1. Sequence analysis of cDNAs derived from RNA transcribed by T7 RNA polymerase in the dual luciferase reporter system, as well as in both a subgenomic replicon and an infectious clone derived from strain JFH1, identified additions and deletions of adenines between codons 8 and 11 due to transcriptional slippage (TS). Moreover, RNA isolated from cells infected with virus generated by JFH1 containing the A-rich tract also contained heterogeneity in the adenine sequence, strongly suggesting TS by the NS5B viral polymerase. These findings have important implications for insight into frameshifting events in HCV-1 and demonstrate for the first time the involvement of transcriptional slippage in this recoding event.

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 MiR-218 inhibits malignant phenotypes of glioma by targeting TNC/AKT/AP-1/TGFβ1 feedback signaling loop

2020 ◽  
Author(s):  
Siwen Dang ◽  
Rui Zhang ◽  
Sijia Tian ◽  
Banjun Ruan ◽  
Peng Hou ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Malignant Tumors ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Normal Brain ◽  
Reporter System ◽  
Control Subjects ◽  
Luciferase Reporter System

Abstract Background: Gliomas are the most common and malignant tumors in the brain of humans, and the prognosis of glioma patient is very poor. MicroRNAs (miRNAs) play critical roles in different types of cancer by regulating gene expression at the posttranscriptional levels. Although miR-218 has been reported to be downregulated in gliomas, its role in gliomas still remains largely unknown. Methods: MiR-218 expression in gliomas and normal brain tissues (control subjects) were analyzed using TCGA dataset. The biological roles of miR-218 in glioma cells were determined by a series of in vitro and in vivo studies. The dual-luciferase reporter system was performed to identify potential targets of miR-218. The regulatory effect of miR-218 on TNC/AKT/AP-1/TGFβ1 pathway was evaluated by dual-luciferase reporter system and western blot.Results: We demonstrated miR-218 was significantly downregulated in gliomas compared to control subjects, and exerted a potent tumor suppressor in glioma cells by inhibiting cell proliferation, colony formation, migration, invasion and tumorigenic potential in nude mice, as well as inducing cell cycle arrest and apoptosis.Mechanistically, miR-218 inhibited malignant phenotypes of glioma cells by binding to the 3’ UTR of its target TNC and subsequently repressing its expression. As a result, it could reduce AKT phosphorylation and subsequently inhibit transcriptional activity of AP-1 by reducing JNK phosphorylation, downregulating the expression of TGFβ1, while TGFβ1 is able to, in turn, activate the TNC/AKT/AP-1 signaling axis.Conclusions: Our data uncover a previously unknown tumor suppressor role of miR-218 in glioma by blocking the TNC/AKT/AP-1/TGFβ1 positive feedback loop.

scholarly journals LncRNA FGD5-AS1 Facilitates the Radioresistance of Breast Cancer Cells by Enhancing MACC1 Expression Through Competitively Sponging miR-497-5p

2021 ◽  
Vol 11 ◽  
Author(s):  
Ji Li ◽  
Changjiang Lei ◽  
Bineng Chen ◽  
Qingfang Zhu
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cell Lines ◽  
Radiation Resistance ◽  
Luciferase Reporter ◽  
Weekly Dose ◽  
Loss Of Function ◽  
Reporter System ◽  
X Ray ◽  
Luciferase Reporter System

BackgroundLncRNA-FGD5-AS1, as an oncogene, participates in the development and progress of various cancers. However, the exact role and the molecular mechanisms by which FGD5-AS1 regulates radiosensitivity in breast cancer (BC) remains largely unknown.MethodsWe used X-Ray weekly-dose-increase method to establish radiation-resistance cell lines. Bioinformatics tools analyze the expression of FGD5-AS1 in breast cancer tissue and evaluated the relationship between FGD5-AS1 and clinic-pathological features. CCK-8 and colony formation were used to analyze cell proliferation. Western blotting and qPCR were applied to detect protein and gene expression, respectively. RNA interference was used to knock down the endogenous gene expression. Luciferase reporter system and immunoprecipitates were applied to verify the target of FGD5-AS1.ResultFGD5-AS1 was overexpressed in BC tissues and radiation-resistance cell lines. Higher levels of FGD5-AS1 predicted poorer clinical characteristics and prognosis. Loss-of-function FGD5-AS1 sensitized BC cells to X-ray, meanwhile, the cell gained radiation-resistance when exogenous FGD5-AS1 was expressed. FGD5-AS1 depletion arrested cells at G0/G1 and triggers cell apoptosis. The starBase database (ENCORI), predicted binding site of miR-497-5p in FGD5-AS1 sequence, and luciferase reporter system and immunoprecipitates verified miR-497-5p was the target of FGD5-AS1. Furthermore, MACC1 was predicted and verified as the target of miR-497-5p. Loss-of-function FGD5-AS1 sensitized ionizing radiation was rescued by the up-regulation of MACC1 and the inhibition of miR-497.ConclusionFGD5-AS1 displays an oncogene profile in CRC; patients with high expression of FGD5-AS1 should benefit less from radiotherapy and need a more frequent follow-up. Besides, FGD5-AS1 may be a potential therapeutic target for CRC.

scholarly journals Elevated microRNA-21 Is a Brake of Inflammation Involved in the Development of Nasal Polyps

2021 ◽  
Vol 12 ◽  
Author(s):  
Ruowu Liu ◽  
Jintao Du ◽  
Jiao Zhou ◽  
Bing Zhong ◽  
Luo Ba ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Ex Vivo ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Nasal Epithelial Cells ◽  
Protein Levels ◽  
Control Subjects ◽  
Luciferase Reporter System ◽  
Suppressive Cytokine

BackgroundCRSwNP is an inflammatory disease but the mechanism is not yet fully understood. MiR-21, a member of miRNAs, has been reported to play roles in mediating inflammation. However, the expression of miR-21 and its role in patients with CRSwNP remain elusive.MethodsTurbinates from control subjects, uncinate processes from CRSsNP, polyp tissues from CRSwNP, and nasal epithelial cells brushed from nasal mucosa were collected. The expression of miR-21 and cytokines in nasal tissues and epithelial cells were detected by qPCR. The localization of miR-21 was detected by ISH, and its target was identified by bioinformation analysis, qPCR, IHC, WB, and luciferase reporter system. The protein and mRNA of PDCD4 and NF-κB P65 were determined by WB and qPCR after miR-21 transfection in HNEpC. The role of miR-21 on cytokines was analyzed in HNEpC and nasal polyp explants.ResultsMiR-21 was upregulated in CRSwNP relative to control subjects by qPCR, which was determined mainly in nasal epithelial cells of CRSwNP by ISH. Both pro-inflammation cytokines (IL-1β, IL-6, IL-8, IL-25, and TSLP) and a suppressive cytokine (IL-10) were overexpressed in the epithelial cells of CRSwNP. The expression of miR-21 was positively correlated with IL-10 and negatively correlated with IL-6, IL-8, IL-33, and TSLP in the epithelial cells of CRSwNP. As a potential target of miR-21, the expression of PDCD4 was negatively correlated with miR-21 in CRSwNP. In HNEpC, miR-21 could reduce the expression of PDCD4 at both mRNA and protein levels, and bioinformation analysis and luciferase reporter system confirmed PDCD4 as one target of miR-21. Furthermore, miR-21 could decrease the activation of NF-κB and increase IL-10 mRNA. Both SEB and LPS could elevate miR-21, with IL-25, IL-33, TSLP induced by SEB and IL-1β, IL-6, IL-8 induced by LPS, while the miR-21 could regulate the expression of IL-33, TSLP, IL-1β, IL- 6 and IL-8 in vitro and ex vivo. Clinically, miR-21 expression was inversely correlated with the Lund-Mackay CT scores and the Lund-Kennedy scores in CRSwNP.ConclusionMiR-21 could be a prominent negative feedback factor in the inflammation process to attenuate the expression of pro-inflammatory cytokines, thereby playing an anti-inflammation role in CRSwNP.

scholarly journals MiR-218 inhibits malignant phenotypes of glioma by targeting TNC/AKT/AP-1/TGFβ1 feedback signaling loop

2020 ◽  
Author(s):  
Siwen Dang ◽  
Rui Zhang ◽  
Sijia Tian ◽  
Banjun Ruan ◽  
Peng Hou ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Regulation Of Gene Expression ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Normal Brain ◽  
Reporter System ◽  
Control Subjects ◽  
Luciferase Reporter System

Abstract Background: Gliomas are the most malignant and common tumors in human brains, and the prognosis of glioma patient is very poor. MicroRNAs (miRNAs) play critical roles in different types of cancer by performing posttranscriptional regulation of gene expression. Although miR-218 has been demonstrated decreased in gliomas, its role in gliomas still remains largely unknown. Methods: MiR-218 expression were analyzed in gliomas and normal brain tissues (control subjects) using TCGA dataset. A series of in vitro and in vivo studies was performed to determine the biological roles of miR-218 in glioma cells. Potential targets of miR-218 were identified using dual-luciferase reporter system. Western blot and dual-luciferase reporter system were performed to evaluate the regulatory effect of miR-218 on TNC/AKT/AP-1/TGFβ1 pathway.Results: We demonstrated miR-218 was significantly downregulated in gliomas compared to control subjects, and played potent tumor suppressor roles in glioma cells by inhibited cell proliferation, colony formation, migration, invasion and tumorigenic potential in nude mice, as well as inducing cell cycle arrest and apoptosis.Mechanistically, miR-218 inhibited malignant phenotypes of glioma cells by binding to the 3’ UTR of its target TNC and subsequently repressing its expression. As a result, it could reduce AKT phosphorylation and subsequently inhibit transcriptional activity of AP-1 by reducing JNK phosphorylation, downregulating the expression of TGFβ1, while TGFβ1 is able to, in turn, activate the TNC/AKT/AP-1 signaling axis.Conclusions: Our data uncover a previously unknown tumor suppressor role of miR-218 by blocking the TNC/AKT/AP-1/TGFβ1 positive feedback loop in glioma.

The use of the luciferase reporter system forin planta gene expression studies

2000 ◽  
Vol 18 (2) ◽  
pp. 143-144 ◽  
Author(s):  
Wessel Van Leeuwen ◽  
Marc J. M. Hagendoorn ◽  
Tom Ruttink ◽  
Remco Van Poecke ◽  
Linus H. W. Van Der Plas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Expression Studies ◽  
Luciferase Reporter System ◽  
Gene Expression Studies

scholarly journals miR-92 regulates the proliferation, migration, invasion and apoptosis of glioma cells by targeting neogenin

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 283-291
Author(s):  
Yi Wang ◽  
Yaohui Tian ◽  
Zonghao Li ◽  
Zhaoke Zheng ◽  
Liangliang Zhu
Keyword(s):  
Cell Proliferation ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter System ◽  
Pathological Mechanism ◽  
Glioma Cell Proliferation ◽  
Luciferase Reporter System ◽  
U87 Cells

AbstractThis study aimed to explore the pathological mechanism in regulating glioma progression. The expression of miR-92 and neogenin was evaluated by qRT-PCR and western blot. Cell viability and apoptosis were measured by MTT and flow cytometry assays, respectively. The migration and invasion abilities were examined by transwell assays. The interaction between miR-92 and neogenin was conducted by dual-luciferase reporter system. As a result, we found that the expression of miR-92 was up-regulated in glioma tissues and cell lines. Down-regulation of miR-92 inhibited glioma cell proliferation, migration, invasion and promoted cell apoptosis rate of U251 and U87 cells. Notably, miR-92 was identified to directly target to 3’-UTR of neogenin. Furthermore, neogenin was down-regulated in glioma tissues and cells in a miR-92-correlated manner. Overexpression of neigenin could cause similar results to miR-92 knockdown in U251 and U87 cells. However, the silencing of neogenin partially reversed the effects of miR-92 knockdown on cell proliferation, migration, invasion and apoptosis of glioma cells in vitro. In conclusion, we clarified that miR-92 knockdown could suppress the malignant progression of glioma cells in vitro by targeting neogenin. Therefore, miR-92 could serve as a potential diagnostic and prognostic marker in glioma patients

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