VARS高表达致缬氨酸代谢失衡促进多发性骨髓瘤细胞生长

High expression of VARS promotes the growth of multiple myeloma cells by causing imbalance in valine metabolism

目的:多发性骨髓瘤(multiple myeloma,MM)是好发于中老年的浆细胞恶性肿瘤,发病机制尚不清楚。由于其多发、复发、耐药的特点,MM仍是一种无法治愈的疾病。氨基酸代谢异常是MM的重要特征之一。氨基酸重要的代谢途径是作为基本原料参与蛋白质合成。氨酰转移核糖核酸合成酶(aminoacyl transfer ribonucleic acid synthetase,ARS)基因是蛋白质合成过程中的关键调节基因。本研究旨在探究在MM中异常表达的氨基酸代谢关键因子ARS通过调控氨基酸代谢影响MM生长的分子机制。方法:对应基因编号合并基因表达综合(Gene Expression Omnibus,GEO)数据库中基因表达谱GSE5900数据集及GSE2658数据集数据,对ARS家族基因表达数据进行标准化处理。采用GSEA_4.2.0软件分析健康供者(healthy donor,HD)与MM患者基因富集的差异。采用GraphPad Prism 7绘制基因热图并进行数据分析。采用Kaplan-Meier及Cox回归模型分别对ARS家族基因表达与MM患者预后情况进行单因素及多因素回归分析。收集7例MM初诊患者的骨髓样本,使用CD138抗体磁珠分选获得CD138+、CD138−细胞,采用real-time RT-PCR分析ARS在MM临床样本中的表达情况。以人B淋巴细胞GM12878细胞,人MM细胞系ARP1、NCI-H929、OCI-MY5、U266、RPMI 8266、OPM-2、JJN-3、KMS11、MM1.s细胞为研究对象,采用real-time RT-PCR及蛋白质印迹法分析ARS在MM细胞系中的表达情况。利用短发夹核糖核酸(short hairpin RNA,shRNA)慢病毒构建基因敲减质粒(VARS-sh组),将其与空载质粒(scramble组)分别转染HEK 293T细胞后进行病毒包装,分别感染ARP1、OCI-MY5细胞并建立稳定表达的细胞系,分别采用细胞计数法和流式细胞术检测敲减缬氨酰tRNA合成酶(valyltRNA synthetase,VARS)基因对MM细胞增殖和凋亡的影响。根据VARS表达将GEO数据分为高表达组和低表达组,通过生物信息学分析探索VARS影响的下游通路,采用飞行时间质谱(gas chromatography time-of-flight/mass spectrometry,GC-TOF/MS)及高效液相色谱(high performance liquid chromatography,HPLC)分别检测临床患者骨髓样本CD138+细胞及敲减VARS基因的ARP1、OCI-MY5细胞及上清液中缬氨酸含量。结果:基因富集分析结果表明:与HD相比,tRNA加工相关的基因在MM中显著富集(P<0.0001)。进一步筛选tRNA加工通路相关子集发现胞质氨酰tRNA合成酶家族基因在MM中显著富集(P<0.0001)。基因表达热图结果表明GEO数据中ARS家族基因除丙氨酰tRNA合成酶(alanyl-tRNA synthetase,AARS)、精氨酰tRNA合成酶(arginyl-tRNA synthetase,RARS)、丝氨酰tRNA合成酶(seryl-tRNA synthetase,SARS)外,其余ARS家族基因均在MM中高表达(均P<0.01),且随着意义未明单克隆丙种球蛋白血症(monoclonal gammopathy of undetermined significance,MGUS)到MM的发展进程,基因表达水平逐渐增加。Kaplan-Meier生存情况单因素分析结果表明甲硫氨酰tRNA合成酶(methionyl-tRNA synthetase,MARS)、天冬酰胺基tRNA合成酶(asparaginyl-tRNA synthetase,NARS)、VARS高表达组与低表达组MM患者预后情况差异均具有统计学意义(均P<0.05)。Cox回归模型多因素分析结果表明:VARS的高表达与MM的总生存时间异常有关(HR=1.83,95%CI 1.10~3.06,P=0.021);NARS(HR=0.90,95%CI 0.34~2.38)及MARS(HR=1.59,95%CI 0.73~3.50)的高表达均对MM患者的总生存时间无影响(均P>0.05)。Real-time RT-PCR及蛋白质印迹法结果表明VARS、MARS和NARS在临床患者CD138+MM细胞及MM细胞系中均高表达(均P<0.05)。细胞计数法及流式细胞术结果表明,敲减VARS的MM细胞增殖能力被显著抑制(P<0.01),细胞凋亡率明显升高(P<0.05)。生物信息学分析结果表明除包括细胞周期在内的通路受VARS调控外,缬氨酸、亮氨酸和异亮氨酸分解代谢通路被上调。非靶向代谢组学数据表明:与HD相比,MM患者的CD138+MM细胞中缬氨酸含量降低(P<0.05)。HPLC结果表明:与scramble组相比,VARS-shRNA组的ARP1细胞与培养基上清液及OCI-MY5培养基上清液中缬氨酸含量均增加(均P<0.05)。结论:VARS在MM中异常高表达,且与MM患者的不良预后相关。VARS可通过影响缬氨酸代谢调控促进MM细胞的生长。

Objective: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.Methods: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.Results: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).Conclusion: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.