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Abstract：
BACKGROUND: A series of studies have demonstrated the emerging involvement of transfer RNA (tRNA) processing during the progression of tumours. Nevertheless, the roles and regulating mechanisms of tRNA processing genes in neuroblastoma (NB), the prevalent malignant tumour outside the brain in children, are yet unknown.
METHODS: Analysis of multi-omics results was conducted to identify crucial regulators of downstream tRNA processing genes. Co-immunoprecipitation and mass spectrometry methods were utilised to measure interaction between proteins. The impact of transcriptional regulators on expression of downstream genes was measured by dual-luciferase reporter, chromatin immunoprecipitation, western blotting and real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) methods. Studies have been conducted to reveal impact and mechanisms of transcriptional regulators on biological processes of NB. Survival differences were analysed using the log-rank test.
RESULTS: c-Myc was identified as a transcription factor driving tRNA processing gene expression and subsequent malate-aspartate shuttle (MAS) in NB cells. Mechanistically, c-Myc directly promoted the expression of glutamyl-prolyl-tRNA synthetase (EPRS) and leucyl-tRNA synthetase (LARS), resulting in translational up-regulation of glutamic-oxaloacetic transaminase 1 (GOT1) as well as malate dehydrogenase 1 (MDH1) via inhibiting general control nonrepressed 2 or activating mechanistic target of rapamycin signalling. Meanwhile, lamin A (LMNA) inhibited c-Myc transactivation via physical interaction, leading to suppression of MAS, aerobic glycolysis, tumourigenesis and aggressiveness. Pre-clinically, lobeline was discovered as a LMNA-binding compound to facilitate its interaction with c-Myc, which inhibited aminoacyl-tRNA synthetase expression, MAS and tumour progression of NB, as well as growth of organoid derived from c-Myc knock-in mice. Low levels of LMNA or elevated expression of c-Myc, EPRS, LARS, GOT1 or MDH1 were linked to a worse outcome and a shorter survival time of clinical NB patients.
CONCLUSIONS: These results suggest that targeting c-Myc transactivation by LMNA inhibits tRNA processing essential for MAS and tumour progression.
METHODS: Analysis of multi-omics results was conducted to identify crucial regulators of downstream tRNA processing genes. Co-immunoprecipitation and mass spectrometry methods were utilised to measure interaction between proteins. The impact of transcriptional regulators on expression of downstream genes was measured by dual-luciferase reporter, chromatin immunoprecipitation, western blotting and real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) methods. Studies have been conducted to reveal impact and mechanisms of transcriptional regulators on biological processes of NB. Survival differences were analysed using the log-rank test.
RESULTS: c-Myc was identified as a transcription factor driving tRNA processing gene expression and subsequent malate-aspartate shuttle (MAS) in NB cells. Mechanistically, c-Myc directly promoted the expression of glutamyl-prolyl-tRNA synthetase (EPRS) and leucyl-tRNA synthetase (LARS), resulting in translational up-regulation of glutamic-oxaloacetic transaminase 1 (GOT1) as well as malate dehydrogenase 1 (MDH1) via inhibiting general control nonrepressed 2 or activating mechanistic target of rapamycin signalling. Meanwhile, lamin A (LMNA) inhibited c-Myc transactivation via physical interaction, leading to suppression of MAS, aerobic glycolysis, tumourigenesis and aggressiveness. Pre-clinically, lobeline was discovered as a LMNA-binding compound to facilitate its interaction with c-Myc, which inhibited aminoacyl-tRNA synthetase expression, MAS and tumour progression of NB, as well as growth of organoid derived from c-Myc knock-in mice. Low levels of LMNA or elevated expression of c-Myc, EPRS, LARS, GOT1 or MDH1 were linked to a worse outcome and a shorter survival time of clinical NB patients.
CONCLUSIONS: These results suggest that targeting c-Myc transactivation by LMNA inhibits tRNA processing essential for MAS and tumour progression.
摘要：
背景：一系列研究表明转移RNA（tRNA）加工在肿瘤进展过程中的出现。然而，tRNA加工基因在神经母细胞瘤（NB）中的作用和调控机制，儿童脑外常见的恶性肿瘤，还未知。
方法：对多组学结果进行分析，以确定下游tRNA加工基因的关键调节因子。使用免疫共沉淀和质谱方法来测量蛋白质之间的相互作用。通过双荧光素酶报告基因检测转录调节因子对下游基因表达的影响,染色质免疫沉淀，蛋白质印迹和实时定量逆转录聚合酶链反应（RT-PCR）方法。已经进行了研究以揭示转录调节因子对NB生物学过程的影响和机制。使用对数秩检验分析生存差异。
结果：c-Myc被鉴定为在NB细胞中驱动tRNA加工基因表达和随后的苹果酸-天冬氨酸穿梭（MAS）的转录因子。机械上,c-Myc直接促进谷氨酰-氨酰-tRNA合成酶（EPRS）和亮氨酰-tRNA合成酶（LARS）的表达，通过抑制一般控制未抑制的2或激活雷帕霉素信号的机制靶标，导致谷氨酸-草酰乙酸转氨酶1（GOT1）和苹果酸脱氢酶1（MDH1）的翻译上调。同时,层粘连蛋白A（LMNA）通过物理相互作用抑制c-Myc反式激活，导致对MAS的压制，有氧糖酵解，肿瘤发生和侵略性。临床前,洛贝林被发现是一种LMNA结合化合物，以促进其与c-Myc的相互作用，抑制氨酰tRNA合成酶的表达，MAS与NB的肿瘤进展，以及来自c-Myc敲入小鼠的类器官的生长。低水平的LMNA或c-Myc表达升高，EPRS,LARS,GOT1或MDH1与临床NB患者的预后较差和生存时间较短有关。
结论：这些结果表明，LMNA靶向c-Myc转录激活抑制了MAS和肿瘤进展所必需的tRNA加工。
方法：对多组学结果进行分析，以确定下游tRNA加工基因的关键调节因子。使用免疫共沉淀和质谱方法来测量蛋白质之间的相互作用。通过双荧光素酶报告基因检测转录调节因子对下游基因表达的影响,染色质免疫沉淀，蛋白质印迹和实时定量逆转录聚合酶链反应（RT-PCR）方法。已经进行了研究以揭示转录调节因子对NB生物学过程的影响和机制。使用对数秩检验分析生存差异。
结果：c-Myc被鉴定为在NB细胞中驱动tRNA加工基因表达和随后的苹果酸-天冬氨酸穿梭（MAS）的转录因子。机械上,c-Myc直接促进谷氨酰-氨酰-tRNA合成酶（EPRS）和亮氨酰-tRNA合成酶（LARS）的表达，通过抑制一般控制未抑制的2或激活雷帕霉素信号的机制靶标，导致谷氨酸-草酰乙酸转氨酶1（GOT1）和苹果酸脱氢酶1（MDH1）的翻译上调。同时,层粘连蛋白A（LMNA）通过物理相互作用抑制c-Myc反式激活，导致对MAS的压制，有氧糖酵解，肿瘤发生和侵略性。临床前,洛贝林被发现是一种LMNA结合化合物，以促进其与c-Myc的相互作用，抑制氨酰tRNA合成酶的表达，MAS与NB的肿瘤进展，以及来自c-Myc敲入小鼠的类器官的生长。低水平的LMNA或c-Myc表达升高，EPRS,LARS,GOT1或MDH1与临床NB患者的预后较差和生存时间较短有关。
结论：这些结果表明，LMNA靶向c-Myc转录激活抑制了MAS和肿瘤进展所必需的tRNA加工。
