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全面转录组分析鉴定出 FXN 和 BDNF 是弗里德里希共济失调患者中 miRNA 的新靶点。

A Comprehensive Transcriptome Analysis Identifies FXN and BDNF as Novel Targets of miRNAs in Friedreich's Ataxia Patients.

机构信息

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

Department of Biochemistry and Molecular Genetics, UAB Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL, USA.

出版信息

Mol Neurobiol. 2020 Jun;57(6):2639-2653. doi: 10.1007/s12035-020-01899-1. Epub 2020 Apr 14.

Abstract

Friedreich's ataxia (FRDA) is a genetic neurodegenerative disease that is caused by guanine-adenine-adenine (GAA) nucleotide repeat expansions in the first intron of the frataxin (FXN) gene. Although present in the intron, this mutation leads to a substantial decrease in protein expression. Currently, no effective treatment is available for FRDA, and, in addition to FXN, other targets with therapeutic potential are continuously sought. As miRNAs can regulate the expression of a broad spectrum of genes, are used as biomarkers, and can serve as therapeutic tools, we decided to identify and characterize differentially expressed miRNAs and their targets in FRDA cells compared to unaffected control (CTRL) cells. In this study, we performed an integrated miRNAseq and RNAseq analysis using the same cohort of primary FRDA and CTRL cells. The results of the transcriptome studies were supported by bioinformatic analyses and validated by qRT-PCR. miRNA interactions with target genes were assessed by luciferase assays, qRT-PCR, and immunoblotting. In silico analysis identified the FXN transcript as a target of five miRNAs upregulated in FRDA cells. Further studies confirmed that miRNA-224-5p indeed targets FXN, resulting in decreases in mRNA and protein levels. We also validated the ability of miRNA-10a-5p to bind and regulate the levels of brain-derived neurotrophic factor (BDNF), an important modulator of neuronal growth. We observed a significant decrease in the levels of miRNA-10a-5p and increase in the levels of BDNF upon correction of FRDA cells via zinc-finger nuclease (ZFN)-mediated excision of expanded GAA repeats. Our comprehensive transcriptome analyses identified miRNA-224-5p and miRNA-10a-5p as negative regulators of the FXN and BDNF expression, respectively. These results emphasize not only the importance of miRNAs in the pathogenesis of FRDA but also their potential as therapeutic targets for this disease.

摘要

弗里德赖希共济失调(FRDA)是一种遗传性神经退行性疾病,由 FXN 基因第一内含子中的鸟嘌呤-腺嘌呤-腺嘌呤(GAA)核苷酸重复扩增引起。尽管这种突变存在于内含子中,但它导致蛋白质表达的显著减少。目前,FRDA 没有有效的治疗方法,除了 FXN 之外,还在不断寻找具有治疗潜力的其他靶点。由于 miRNA 可以调节广泛的基因表达,可用作生物标志物,并可作为治疗工具,我们决定鉴定和表征 FRDA 细胞与未受影响的对照(CTRL)细胞相比差异表达的 miRNA 及其靶标。在这项研究中,我们使用相同的 FRDA 和 CTRL 细胞队列进行了 miRNAseq 和 RNAseq 分析。转录组研究的结果得到了生物信息学分析的支持,并通过 qRT-PCR 进行了验证。miRNA 与靶基因的相互作用通过荧光素酶测定、qRT-PCR 和免疫印迹进行评估。计算机分析确定 FXN 转录物是 FRDA 细胞中上调的五个 miRNA 的靶标。进一步的研究证实,miRNA-224-5p 确实靶向 FXN,导致 mRNA 和蛋白水平降低。我们还验证了 miRNA-10a-5p 结合和调节脑源性神经营养因子(BDNF)水平的能力,BDNF 是神经元生长的重要调节剂。我们观察到通过锌指核酸酶(ZFN)介导的扩增 GAA 重复序列切除校正 FRDA 细胞后,miRNA-10a-5p 的水平显著降低,BDNF 的水平升高。我们的综合转录组分析确定 miRNA-224-5p 和 miRNA-10a-5p 分别为 FXN 和 BDNF 表达的负调节剂。这些结果不仅强调了 miRNA 在 FRDA 发病机制中的重要性,还强调了它们作为该疾病治疗靶点的潜力。

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