多发性骨髓瘤（MM）是一种发生在中老年的浆细胞恶性肿瘤。由于频繁复发和药物耐药性，MM仍然是一种不可治愈的疾病。然而，其发病机制仍然不清楚。异常的氨基酸代谢是MM的重要特征之一，重要的氨基酸代谢途径参与了蛋白质合成的基本原料。氨酰转移核酸合成酶（ARS）基因是蛋白质合成中的关键调节基因。本研究旨在探索ARS作为氨基酸代谢关键因子对MM中氨基酸代谢的调控机制以及对MM生长的影响的分子机制。将对应的基因编号与Gene Expression Omnibus (GEO)数据库中的基因表达谱GSE5900数据集和GSE2658数据集结合，标准化ARS的基因表达数据。利用GSEA_4.2.0软件分析GEO数据库中健康供体（HD）和MM患者之间基因富集的差异。使用GraphPad Prism 7绘制热图并进行数据分析。分别使用Kaplan-Meier和Cox回归模型分析ARS基因的表达和MM患者的预后。收集7例新诊断的MM患者的骨髓样本，使用CD138抗体磁珠获得CD138+和CD138-细胞，并通过实时RT-PCR分析MM临床样本中ARS的表达。选择人类B淋巴细胞GM12878细胞和人类MM细胞系ARP1, NCI-H929, OCI-MY5, U266, RPMI 8266, OPM-2, JJN-3, KMS11和MM1.s细胞作为研究对象。通过实时RT-PCR和Western blot分析MM细胞系中ARS的表达。利用短发夹RNA (shRNA) 病毒构建基因敲除质粒（VARS-sh组）。分别将空载体质粒（scramble组）和基因敲除质粒（VARS-sh组）转染入HEK 293T细胞进行病毒包装。通过感染ARP1和OCI-MY5细胞建立稳定表达细胞系，并通过细胞计数和流式细胞术测定敲除valyl-tRNA合成酶（VARS）基因对MM细胞增殖和凋亡的影响。根据VARS的表达将GEO数据分为高表达组和低表达组。进行生物信息学分析以探索受VARS影响的下游通路。利用气相色谱-飞行时间质谱法（GC-TOF/MS）和高效液相色谱法（HPLC）检测CD138+细胞和骨髓样本中敲除VARS基因的ARP1、OCI-MY5细胞以及上清液中的缬氨酸含量。基因富集分析显示与HD相比，MM中tRNA加工相关基因明显富集（P<0.0001）。进一步筛选与tRNA处理通路相关的亚组，发现细胞质氨酰-tRNA合成酶家族基因在MM中显著富集（P<0.0001）。基因表达热图结果显示，除丙氨酰-tRNA合成酶（AARS）、精氨酰-tRNA合成酶（RARS）、丝氨酰-tRNA合成酶（SARS）外，GEO数据中ARS家族基因在MM中高度表达（所有P<0.01）。随着年龄从单克隆酸病变（MGUS）演变为MM，基因表达水平逐渐增加。Kaplan-Meier单因素分析生存结果显示高表达组和低表达组之间MM患者的预后有显著差异的亚组中，甲硫酰-tRNA合成酶（MARS）、天门冬氨酸-tRNA合成酶（NARS）和VARS（所有P<0.05）的表达之间存在显著差异。Cox回归模型多因素分析显示VARS的高表达与MM整体生存时间异常相关（HR=1.83，95% CI 1.10 to 3.06，P=0.021）。NARS的高表达（HR=0.90，95% CI 0.34 to 2.38）和MARS的高表达（HR=1.59，95% CI 0.73 to 3.50）都对MM患者的整体生存时间没有影响（两者P>0.05）。实时RT-PCR和Western blot结果显示VARS、MARS和NARS在CD138+ MM细胞和临床患者MM细胞系中高表达（所有P<0.05）。细胞计数和流式细胞术结果显示敲除VARS显著抑制MM细胞的增殖（P<0.01），增加凋亡比例（P<0.05）。生物信息学分析显示除VARS调控的细胞周期等几个通路外，缬、亮和异亮氨酸降解通路上调。非靶向代谢组学数据显示与HD相比，MM患者的CD138+肿瘤细胞中缬氨酸含量减少（P<0.05）。HPLC结果显示与scramble组相比，VARS-shRNA组的ARP1细胞和OCI-MY5细胞中细胞内和培养上清液中缬氨酸含量增加（所有P<0.05）。表达VARS异常高的MM患者预后不良。VARS通过影响缬氨酸代谢的调节促进了MM细胞的恶性生长。

Multiple myeloma (MM) is a plasma cell malignancy occurring in middle and old age. MM is still an incurable disease due to its frequent recurrence and drug resistance. However, its pathogenesis is still unclear. Abnormal amino acid metabolism is one of the important characteristics of MM, and the important metabolic pathway of amino acids participates in protein synthesis as basic raw materials. Aminoacyl transfer ribonucleic acid synthetase (ARS) gene is a key regulatory gene in protein synthesis. This study aims to explore the molecular mechanism for ARS, a key factor of amino acid metabolism, in regulating amino acid metabolism in MM and affecting MM growth.The corresponding gene number was combined with the gene expression profile GSE5900 dataset and GSE2658 dataset in Gene Expression Omnibus (GEO) database to standardize the gene expression data of ARS. GSEA_4.2.0 software was used to analyze the difference of gene enrichment between healthy donors (HD) and MM patients in GEO database. GraphPad Prism 7 was used to draw heat maps and perform data analysis. Kaplan-Meier and Cox regression model were used to analyze the expression of ARS gene and the prognosis of MM patients, respectively. Bone marrow samples from 7 newly diagnosed MM patients were collected, CD138+ and CD138- cells were obtained by using CD138 antibody magnetic beads, and the expression of ARS in MM clinical samples was analyzed by real-time RT-PCR. Human B lymphocyte GM12878 cells and human MM cell lines ARP1, NCI-H929, OCI-MY5, U266, RPMI 8266, OPM-2, JJN-3, KMS11, MM1.s cells were selected as the study objects. The expression of ARS in MM cell lines was analyzed by real-time RT-PCR and Western blotting. Short hairpin RNA (shRNA) lentiviruses were used to construct gene knock-out plasmids (VARS-sh group). No-load plasmids (scramble group) and gene knock-out plasmids (VARS-sh group) were transfected into HEK 293T cells with for virus packaging, respectively. Stable expression cell lines were established by infecting ARP1 and OCI-MY5 cells, and the effects of knockout valyl-tRNA synthetase (VARS) gene on proliferation and apoptosis of MM cells were detected by cell counting and flow cytometry, respectively. GEO data were divided into a high expression group and a low expression group according to the expression of VARS. Bioinformatics analysis was performed to explore the downstream pathways affected by VARS. Gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) and high performance liquid chromatography (HPLC) were used to detect the valine content in CD138+ cells and ARP1, OCI-MY5 cells and supernatant of knockdown VARS gene in bone marrow samples from patients, respectively.Gene enrichment analysis showed that tRNA processing related genes were significantly enriched in MM compared with HD (P<0.0001). Further screening of tRNA processing-pathway related subsets revealed that cytoplasmic aminoacyl tRNA synthetase family genes were significantly enriched in MM (P<0.0001). The results of gene expression heat map showed that the ARS family genes except alanyl-tRNA synthetase (AARS), arginyl-tRNA synthetase (RARS), seryl-tRNA synthetase (SARS) in GEO data were highly expressed in MM (all P<0.01). With the development of monoclonal gammopathy of undetermined significance (MGUS) to MM, the gene expression level was increased gradually. Kaplan-Meier univariate analysis of survival results showed that there were significant differences in the prognosis of MM patients in methionyl-tRNA synthetase (MARS), asparaginyl-tRNA synthetase (NARS) and VARS between the high expression group and the low expression group (all P<0.05). Cox regression model multivariate analysis showed that the high expression of VARS was associated with abnormal overall survival time of MM (HR=1.83, 95% CI 1.10 to 3.06, P=0.021). The high expression of NARS (HR=0.90, 95% CI 0.34 to 2.38) and MARS (HR=1.59, 95% CI 0.73 to 3.50) had no effect on the overall survival time of MM patients (both P>0.05). Real-time RT-PCR and Western blotting showed that VARS, MARS and NARS were highly expressed in CD138+ MM cells and MM cell lines of clinical patients (all P<0.05). Cell counting and flow cytometry results showed that the proliferation of MM cells by knockout VARS was significantly inhibited (P<0.01), the proportion of apoptosis was significantly increased (P<0.05). Bioinformatics analysis showed that in addition to several pathways including the cell cycle regulated by VARS, the valine, leucine and isoleucine catabolic pathways were upregulated. Non-targeted metabolomics data showed reduced valine content in CD138+ tumor cells in MM patients compared to HD (P<0.05). HPLC results showed that compared with the scramble group, the intracellular and medium supernatant content of ARP1 cells and the medium supernatant of OCI-MY5 in the VARS-shRNA group was increased (all P<0.05).MM patients with abnormal high expression of VARS have a poor prognosis. VARS promotes the malignant growth of MM cells by affecting the regulation of valine metabolism.