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RNA-seq analysis reveals significant transcriptome changes in huntingtin-null human neuroblastoma cells
BMC Medical Genomics ( IF 2.1 ) Pub Date : 2021-07-02 , DOI: 10.1186/s12920-021-01022-w
Johanna Bensalel 1 , Hongyuan Xu 1 , Michael L Lu 1 , Enrico Capobianco 2 , Jianning Wei 1
Affiliation  

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.

中文翻译:


RNA-seq分析揭示亨廷顿蛋白缺失的人神经母细胞瘤细胞中转录组的显着变化



亨廷顿蛋白 (Htt) 是亨廷顿病 (HD) 基因突变的产物,亨廷顿病是一种致命的常染色体显性神经退行性疾病。正常的 Htt 对于早期胚胎发生和中枢神经系统的发育至关重要。然而,Htt 在成人组织中的作用尚不明确。最近进行了一项颇有前景的临床试验,其中正常和突变的 Htt mRNA 在 HD 患者中均被敲除,因此迫切需要充分了解在成人组织中敲除/敲低 Htt 的分子后果。 Htt 已被确定为重要的转录调节因子。利用 RNA 测序 (RNA-Seq) 对 HD 的多种细胞类型的转录组变化进行了公正的研究,进一步证实转录失调是 HD 的核心致病机制。然而,目前还缺乏对正常 Htt 转录调控的直接了解。为了研究正常 Htt 的转录作用,我们首先使用 CRISPR(成簇规则间隔短回文重复序列)-Cas9(CRISPR 相关蛋白 9)基因编辑方法敲除人神经母细胞瘤 SH-SY5Y 细胞系中的 Htt。然后,我们对 Htt 缺失和野生型 SH-SY5Y 细胞进行 RNA 序列分析,以探测 Htt 缺失引起的全局转录组变化。一般来说,Htt 对基因转录具有广泛的影响。使用各种生物信息学工具对差异表达基因(DEG)进行功能分析揭示了与细胞通讯和信号传导相关的通路的不规则性,更具体地说,与神经元发育、神经传递和突触信号传导相关的通路的不规则性。我们进一步研究了可能调节这些差异表达基因的转录因子。 与细胞发育相关的被破坏的途径一致,我们发现 Htt 缺失细胞比野生型细胞表现出更慢的细胞增殖。我们最终通过定量 RT-PCR 验证了一些顶级 DEGS。 Htt 缺失细胞中广泛的转录组变化可能是由 Htt 介导的转录调控丧失直接引起的,或者是由于基因调控网络破坏的继发后果。因此,我们的研究提供了与 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 细胞功能的分子机制的理解。
更新日期:2021-07-02
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