MicroRNA-376b-3p Suppresses Choroidal Neovascularization by Regulating Glutaminolysis in Endothelial Cells
Yifan FengLiyang WangChunqiong DongXi YangJing WangXi ZhangYuanzhi YuanJinhui DaiJinhai HuangFei Yuan
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Purpose: Choroidal neovascularization (CNV) is a common pathological change of various ocular diseases that causes serious damage to central vision. Accumulated evidence shows that microRNAs (miRNAs) are closely related with the regulation of endothelial metabolism, which plays crucial roles in angiogenesis. Here, we investigate the molecular mechanism underlying the regulation of endothelial glutamine metabolism by miR-376b-3p in the progression of CNV. Methods: Human retinal microvascular endothelial cells (HRMECs) were transfected with control or miR-376b-3p mimics, and the expression of glutaminase 1 (GLS1), a rate-limiting enzyme in glutaminolysis, was detected by real-time PCR or Western blotting. The biological function and glutamine metabolism of transfected HRMECs were measured by related kits. Luciferase reporter assays were used to validate the CCAAT/enhancer-binding protein beta (CEBPB) was a target of miR-376b-3p. Chromatin immunoprecipitation and RNA immunoprecipitation assays were performed to verify the binding of CEBPB on the promoter region of GLS1. Fundus fluorescein angiography and immunofluorescence detected the effect of miR-376b-3p agomir on rat laser-induced CNV. Results: The expression of miR-376b-3p was decreased, whereas GLS1 expression was increased in the retinal pigment epithelial–choroidal complexes of rats with CNV. HRMECs transfected with miR-376b-3p mimic showed inhibition of CEBPB, resulting in the inactivation of GLS1 transcription and glutaminolysis. Moreover, the miR-376b-3p mimic inhibited proliferation, migration and tube formation but promoted apoptosis in HRMECs, whereas these effects counteracted by α-ketoglutarate supplementation or transfection with CEBPB overexpression plasmid. Finally, the intravitreal administration of the miR-376b-3p agomir restrained CNV formation. Conclusions: Collectively, miR-376b-3p is a suppressor of glutamine metabolism in endothelial cells that could be expected to become a therapeutic target for the treatment of CNV-related diseases.Keywords:
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The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro and in vivo. Firefly luciferase oxidates its substrate, luciferin, resulting in the emission of light. A previous study suggests that the firefly luciferase complementation assay has different luminescence kinetics from full length luciferase. The mechanism behind this is still unknown. Although half of the previously published studies utilizing the firefly luciferase complementation assay consider it quantitative. To understand how the molecular reactions and the changes in the affinity of the protein pair affect experimental results, a mathematical model was constructed. This suggests that previously published studies should be considered qualitative, unless an additional experiment is performed. This new model demonstrates that the luminescence measured is not linearly correlated with the affinity of the protein pair. The model is then used to design a new experiment which allows the firefly luciferase complementation assay to be used quantitatively to detect changes of affinity.
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To aid in the interpretation of high-throughput screening (HTS) results derived from luciferase-based assays, we used quantitative HTS, an approach that defines the concentration−response behavior of each library sample, to profile the ATP-dependent luciferase from Photinus pyralis against more than 70 000 samples. We found that approximately 3% of the library was active, containing only compounds with inhibitory concentration−responses, of which 681 (0.9%) exhibited IC50 < 10 µM. Representative compounds were shown to inhibit purified P. pyralis as well as several commercial luciferase-based detection reagents but were found to be largely inactive against Renilla reniformis luciferase. Light attenuation by the samples was also examined and found to be more prominent in the blue-shifted bioluminescence produced by R. reniformis luciferase than in the bioluminescence produced by P. pyralis luciferase. We describe the structure−activity relationship of the luciferase inhibitors and discuss the use of this data in the interpretation of HTS results and configuration of luciferase-based assays.
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Bioluminescence of the medusa Periphylla is based on the oxidation of coelenterazine catalyzed by luciferase. Periphylla has two types of luciferase: the soluble form luciferase L, which causes the exumbrellar bioluminescence display of the medusa, and the insoluble aggregated form, which is stored as particulate material in the ovary, in an amount over 100 times that of luciferase L. The eggs are especially rich in the insoluble luciferase, which drastically decreases upon fertilization. The insoluble form could be solubilized by 2-mercaptoethanol, yielding a mixture of luciferase oligomers with molecular masses in multiples of approximately 20 kDa. Those having the molecular masses of 20 kDa, 40 kDa, and 80 kDa were isolated and designated, respectively, as luciferase A, luciferase B, and luciferase C. The luminescence activities of Periphylla luciferases A, B, and C were 1.2∼4.1 × 1016 photon/mg · s, significantly higher than any coelenterazine luciferase known, and the quantum yields of coelenterazine catalyzed by these luciferases (about 0.30 at 24 °C) are comparable to that catalyzed by Oplophorus luciferase (0.34 at 22 °C), which has been considered the most efficient coelenterazine luciferase until now. Luciferase L (32 kDa) could also be split by 2-mercaptoethanol into luciferase A and an accessory protein (approx. 12 kDa), as yet uncharacterized. Luciferases A, B, and C are highly resistant to inactivation: their luminescence activities are only slightly diminished at pH 1 and pH 11 and are enhanced in the presence of 1∼2 M guanidine hydrochloride; but they are less stable to heating than luciferase L, which is practically unaffected by boiling.
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Biochemical properties, spectral parameters of bioluminescence and reaction kinetics for Luciola mingrelica firefly luciferase are described and analysed. The kinetic scheme of the enzymatic process is proposed and discussed. Allosteric regulation of luciferase activity by ATP and its analogues is considered and binding Mg2+ to luciferase shown to increase its activity. Regulation mechanism of luciferase activity by phospholipids is analysed and choline-containing phospholipids shown to be specific luciferase activators. Some properties of firefly luciferase and the luciferase synthesized during firefly mRNA translation in frog oocytes are compared.
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We constructed a novel bacterial genome detection system using zinc finger protein (ZF) fused with firefly luciferase (ZF-luciferase). Taking advantage of the direct recognition of double-stranded DNA (dsDNA) by ZF, we previously constructed bacterial genome detection systems that did not require dehybridization processes. To detect polymerase chain reaction (PCR) products rapidly and with a high sensitivity, we constructed two kinds of ZF-luciferase, Sp1-fused luciferase (Sp1-luciferase), and Zif268-fused luciferase (Zif268-luciferase). ZF-luciferase not only maintains luciferase activity but also shows dsDNA-binding ability and specificity. Furthermore, we succeeded in the detection of 10 copies of the genome of Legionella pneumophila and Escherichia coli O157. ZF-luciferase would be a useful tool for highly sensitive detection of pathogenic bacterial genome.
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Luciferase comprises a family of enzymes that catalyzes luciferin or aliphatic aldehydes oxidation-luminescence.Luciferase genes have been extracted from bacteria,firefly,Renilla,ect.With studying deeply,luciferase is applied to molecular biology,medicine,analysis of microorganisms and other fields.The species,structure and applications of luciferase were researched.
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Background: The firefly luciferase reporter protein is a crucial tool for studies targeting a broad range of biological questions. Importantly, luciferase assays are also widely used to explore mechanisms underlying thyroid hormone dependent regulation of gene expression. However, it was demonstrated that the firefly luciferase reporter is subject to triiodothyronine (T3)-evoked, promoter independent downregulation that is mediated by the thyroid hormone receptor. Since this effect can interfere with readout accuracy, the study aimed to find luciferase reporters that are not susceptible to this phenomenon. Methods: Luciferase reporter constructs were generated under the control of a minimal thymidine kinase (TK) promoter and transiently transfected into JEG-3 cells to test their activity upon T3 treatment. Results: Activity of the TK-(dCpG)Luc encoding a synthetic (dCpG)Luciferase and TK-NanoLuc expressing the NanoLuc reporter was not significantly changed by T3 treatment while the firefly luciferase control was suppressed by ∼2.6-fold. T3 also downregulated the activity of Renilla luciferase by ∼30%. Conclusions: Novel types of luciferase reporters, especially the synthetic (dCpG)Luciferase, can be more accurate to study T3-regulated gene expression than the classical firefly luciferase reporter. Renilla luciferase, a popular transfection control of dual luciferase assays, should be used with caution in conditions with T3 treatment.
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