Department of Oncology, Institute of Clinical Sciences, Sahlgrenska Cancer Center, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
Cancer Rep (Hoboken). 2019 Oct;2(5):e1204. doi: 10.1002/cnr2.1204. Epub 2019 Jul 25.
The human orphan receptor TLX (NR2E1) is a key regulator of neurogenesis, adult stem cell maintenance, and tumorigenesis. However, little is known about the genetic and transcriptomic events that occur following TLX overexpression in human cell lines.
Here, we used cytogenetics and RNA sequencing to investigate the effect of TLX overexpression with an inducible vector system in the HEK 293T cell line.
Conventional spectral karyotyping was used to identify chromosomal abnormalities, followed by fluorescence in situ hybridization (FISH) analysis on chromosome spreads to assess TLX DNA copy number. Illumina paired-end whole transcriptome sequencing was then performed to characterize recurrent genetic variants (single nucleotide polymorphisms (SNPs) and indels), expressed gene fusions, and gene expression profiles. Lastly, flow cytometry was used to analyze cell cycle distribution. Intriguingly, we show that upon transfection with a vector containing the human TLX gene (eGFP-hTLX), an isochromosome forms on the long arm of chromosome 6, thereby resulting in DNA gain of the TLX locus (6q21) and upregulation of TLX. Induction of the eGFP-hTLX vector further increased TLX expression levels, leading to G0-G1 cell cycle arrest, genetic aberrations, modulation of gene expression patterns, and crosstalk with other nuclear receptors (AR, ESR1, ESR2, NR1H4, and NR3C2). We identified a 49-gene signature associated with central nervous system (CNS) development and carcinogenesis, in addition to potentially cancer-driving gene fusions (LARP1-CNOT8 and NSL1-ZDBF2) and deleterious genetic variants (frameshift insertions in the CTSH, DBF4, POSTN, and WDR78 genes).
Taken together, these findings illustrate that TLX may play a pivotal role in tumorigenesis via genomic instability and perturbation of cancer-related processes.
人类孤儿受体 TLX（NR2E1）是神经发生、成人干细胞维持和肿瘤发生的关键调节因子。然而，对于 TLX 在人细胞系中过表达后发生的遗传和转录组事件知之甚少。
本研究采用细胞遗传学和 RNA 测序技术，研究诱导型载体系统中 TLX 过表达对 HEK 293T 细胞系的影响。
采用常规光谱核型分析鉴定染色体异常，随后对染色体展开进行荧光原位杂交（FISH）分析，以评估 TLX DNA 拷贝数。然后进行 Illumina 配对末端全转录组测序，以描述反复出现的遗传变异（单核苷酸多态性（SNP）和插入缺失）、表达基因融合和基因表达谱。最后，通过流式细胞术分析细胞周期分布。有趣的是，我们发现，在用含有人类 TLX 基因的载体（eGFP-hTLX）转染后，6 号染色体长臂上形成一条等臂染色体，从而导致 TLX 基因座（6q21）的 DNA 获得和 TLX 的上调。eGFP-hTLX 载体的诱导进一步增加了 TLX 的表达水平，导致 G0-G1 细胞周期停滞，遗传异常，基因表达模式的调节，以及与其他核受体（AR、ESR1、ESR2、NR1H4 和 NR3C2）的串扰。我们鉴定了一个与中枢神经系统（CNS）发育和癌变相关的 49 基因特征，以及潜在的致癌基因融合（LARP1-CNOT8 和 NSL1-ZDBF2）和有害的遗传变异（CTSH、DBF4、POSTN 和 WDR78 基因中的框移插入）。
综上所述，这些发现表明 TLX 可能通过基因组不稳定性和干扰与癌症相关的过程在肿瘤发生中发挥关键作用。
