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首页> 外文期刊>BMC Medical Genomics >RNA-seq analysis reveals significant transcriptome changes in huntingtin-null human neuroblastoma cells
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RNA-seq analysis reveals significant transcriptome changes in huntingtin-null human neuroblastoma cells

机译：RNA-SEQ分析揭示了亨廷顿 - 零人神经母细胞瘤细胞的显着转录组变化

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Huntingtin (Htt) protein is the product of the gene mutated in Huntington’s disease (HD), a fatal, autosomal dominant, neurodegenerative disorder. Normal Htt is essential for early embryogenesis and the development of the central nervous system. However, the role of Htt in adult tissues is less defined. Following the recent promising clinical trial in which both normal and mutant Htt mRNA were knocked down in HD patients, there is an urgent need to fully understand the molecular consequences of knocking out/down Htt in adult tissues. Htt has been identified as an important transcriptional regulator. Unbiased investigations of transcriptome changes with RNA-sequencing (RNA-Seq) have been done in multiple cell types in HD, further confirming that transcriptional dysregulation is a central pathogenic mechanism in HD. However, there is lack of direct understanding of the transcriptional regulation by normal Htt. To investigate the transcriptional role of normal Htt, we first knocked out Htt in the human neuroblastoma SH-SY5Y cell line using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) gene editing approach. We then performed RNA-seq analysis on Htt-null and wild type SH-SY5Y cells to probe the global transcriptome changes induced by Htt deletion. In general, Htt has a widespread effect on gene transcription. Functional analysis of the differentially expressed genes (DEGs) using various bioinformatic tools revealed irregularities in pathways related to cell communication and signaling, and more specifically those related to neuron development, neurotransmission and synaptic signaling. We further examined the transcription factors that may regulate these DEGs. Consistent with the disrupted pathways associated with cellular development, we showed that Htt-null cells exhibited slower cell proliferation than wild type cells. We finally validated some of the top DEGS with quantitative RT-PCR. The widespread transcriptome changes in Htt-null cells could be directly caused by the loss of Htt-mediated transcriptional regulation or due to the secondary consequences of disruption in the gene regulatory network. Our study therefore provides valuable information about key genes associated with Htt-mediated transcription and improves our understanding of the molecular mechanisms underlying the cellular functions of normal and mutant Htt.
机译：亨廷顿（HTT）蛋白质是亨廷顿疾病（HD），致命，常染色体显性，神经变性疾病的基因的产物。正常HTT对于早期胚胎发生和中枢神经系统的发展至关重要。然而，定义了HTT在成人组织中的作用。在最近在高清患者中撞击正常和突变体HTT mRNA的有前途的临床试验之后，迫切需要充分了解在成人组织中敲出/下HTT的分子后果。 HTT已被识别为重要的转录调节因子。在HD中的多个细胞类型中已经在多种细胞类型中进行了不偏析转录组的变化（RNA-SEQ），进一步证实转录失调是HD中的中心致病机制。然而，缺乏正常HTT对转录调控的直接理解。为了探讨正常HTT的转录作用，我们首先使用CRISPR（Classed Trangerspaced短语重复）-CAS9（CRISPR相关蛋白质9）基因编辑方法将HTT敲除了人类神经母细胞瘤SH-SEN-SEN-SEC-SENY5Y细胞系中的HTT。然后我们对HTT-NULL和野生型SH-SY5Y细胞进行RNA-SEQ分析，以探测HTT删除引起的全局转录组变化。通常，HTT对基因转录具有广泛的影响。使用各种生物信息工具的差异表达基因（DEGS）的功能分析揭示了与细胞通信和信号传导相关的途径的不规则性，更具体地与神经元发育，神经递质和突触信号相关的途径。我们进一步检查了可能调节这些次数的转录因子。与与细胞发育相关的破坏途径一致，我们表明HTT-NULL细胞表现出比野生型细胞的较慢细胞增殖。我们最终通过定量RT-PCR验证了一些顶部。 HTT-NULL细胞的广泛转录组变化可以直接由HTT介导的转录调节丧失或由于基因调节网络中断的二次后果。因此，我们的研究提供了有关与HTT介导的转录相关的关键基因的有价值的信息，并提高了我们对正常和突变HTT细胞功能下面的分子机制的理解。

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