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Over-expression of microRNA-1 causes arrhythmia by disturbing intracellular trafficking system.

ABSTRACT: Dysregulation of intracellular trafficking system plays a fundamental role in the progression of cardiovascular disease. Up-regulation of miR-1 contributes to arrhythmia, we sought to elucidate whether intracellular trafficking contributes to miR-1-driven arrhythmia. By performing microarray analyses of the transcriptome in the cardiomyocytes-specific over-expression of microRNA-1 (miR-1?Tg) mice and the WT mice, we found that these differentially expressed genes in miR-1?Tg mice were significantly enrichment with the trafficking-related biological processes, such as regulation of calcium ion transport. Also, the qRT-PCR and western blot results validated that Stx6, Braf, Ube3a, Mapk8ip3, Ap1s1, Ccz1 and Gja1, which are the trafficking-related genes, were significantly down-regulated in the miR-1?Tg mice. Moreover, we found that Stx6 was decreased in the heart of mice after myocardial infarction and in the hypoxic cardiomyocytes, and further confirmed that Stx6 is a target of miR-1. Meanwhile, knockdown of Stx6 in cardiomyocytes resulted in the impairments of PLM and L-type calcium channel, which leads to the increased resting ([Ca2+]i). On the contrary, overexpression of Stx6 attenuated the impairments of miR-1 or hypoxia on PLM and L-type calcium channel. Thus, our studies reveals that trafficking-related gene Stx6 may regulate intracellular calcium and is involved in the occurrence of cardiac arrhythmia, which provides new insights in that miR-1 participates in arrhythmia by regulating the trafficking-related genes and pathway.

SUBMITTER: Su X

PROVIDER: S-EPMC5387686 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Over-expression of microRNA-1 causes arrhythmia by disturbing intracellular trafficking system.

Su Xiaomin X Liang Haihai H Wang He H Chen Guizhi G Jiang Hua H Wu Qiuxia Q Liu Tianyi T Liu Qiushuang Q Yu Tong T Gu Yunyan Y Yang Baofeng B Shan Hongli H

Scientific reports 20170411

Dysregulation of intracellular trafficking system plays a fundamental role in the progression of cardiovascular disease. Up-regulation of miR-1 contributes to arrhythmia, we sought to elucidate whether intracellular trafficking contributes to miR-1-driven arrhythmia. By performing microarray analyses of the transcriptome in the cardiomyocytes-specific over-expression of microRNA-1 (miR-1 Tg) mice and the WT mice, we found that these differentially expressed genes in miR-1 Tg mice were significan ...[more]

PMID: 28397788

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Project description:BackgroundA tool for stoichiometric co-expression of effector and target proteins to study intracellular protein trafficking processes has been provided by the so called 2A peptide technology. In this system, the 16-20 amino acid 2A peptide from RNA viruses allows synthesis of multiple gene products from single transcripts. However, so far the use of the 2A technology in plant systems has been limited.Methodology/principal findingsThe aim of this work was to assess the suitability of the 2A peptide technology to study the effects exerted by dominant mutant forms of three small GTPase proteins, RABD2a, SAR1, and ARF1 on intracellular protein trafficking in plant cells. Special emphasis was given to CAH1 protein from Arabidopsis, which is trafficking to the chloroplast via a poorly characterized endoplasmic reticulum-to-Golgi pathway. Dominant negative mutants for these GTPases were co-expressed with fluorescent marker proteins as polyproteins separated by a 20 residue self-cleaving 2A peptide. Cleavage efficiency analysis of the generated polyproteins showed that functionality of the 2A peptide was influenced by several factors. This enabled us to design constructs with greatly increased cleavage efficiency compared to previous studies. The dominant negative GTPase variants resulting from cleavage of these 2A peptide constructs were found to be stable and active, and were successfully used to study the inhibitory effect on trafficking of the N-glycosylated CAH1 protein through the endomembrane system.Conclusions/significanceWe demonstrate that the 2A peptide is a suitable tool when studying plant intracellular protein trafficking and that transient protoplast and in planta expression of mutant forms of SAR1 and RABD2a disrupts CAH1 trafficking. Similarly, expression of dominant ARF1 mutants also caused inhibition of CAH1 trafficking to a different extent. These results indicate that early trafficking of the plastid glycoprotein CAH1 depends on canonical vesicular transport mechanisms operating between the endoplasmic reticulum and Golgi apparatus.

Project description:IntroductionThe increasing morbidity of fungal infections and the prevalence of drug resistance highlighted the discovery of novel antifungal agents and investigation of their modes of action. Iron chelators have been used to treat superficial fungal infections or potentiate the efficacy of certain antifungal drugs. Hinokitiol exhibits potent antifungal activity and iron-chelating ability. However, their relationships have not been established.ObjectivesThis study aims to explore the selectivity of hinokitiol against fungal cells and mammalian cells and determine the role of iron-chelating for the antifungal activity of hinokitiol.MethodsIron probe FeRhonox-1 was used to determine intracellular Fe2+ content. 5-Cyano-2,3-ditolyl tetrazolium chloride probe and Cell Counting Kit-8 were used to detect the mitochondrial respiratory activities. Quantitative real-time PCR and rescue experiments were performed to determine the effect of iron on the antifungal activity of hinokitiol. The effects of hinokitiol on fungal mitochondria were further evaluated using reactive oxygen species probes and several commercial Assay Kits. The ability of hinokitiol to induce resistance in Candida species was carried out using a serial passage method. The in vivo therapeutic effect of hinokitiol was evaluated using Galleria mellonella as an infectious model.ResultsHinokitiol was effective against a panel of Candida strains with multiple azole-resistant mechanisms and persistently inhibited Candida albicans growth. Mechanism investigations revealed that hinokitiol chelated fungal intracellular iron and inhibited the respiration of fungal cells but had minor effects on mammalian cells. Hinokitiol further inhibited the activities of mitochondrial respiratory chain complexes I and II and reduced mitochondrial membrane potential, thereby decreasing intracellular ATP synthesis and increasing detrimental intracellular reductive stress. Moreover, hinokitiol exhibited low potential for inducing resistance in several Candida species and greatly improved the survival of Candida-infected Galleria mellonella.ConclusionsThese findings suggested the potential application of hinokitiol as an iron chelator to treat fungal infections.

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Over-expression of microRNA-1 causes arrhythmia by disturbing intracellular trafficking system.

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Over-expression of microRNA-1 causes arrhythmia by disturbing intracellular trafficking system.

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