核糖体生物合成因子BMS1对神经母细胞瘤细胞增殖的影响  

Effect of the ribosome biogenesis factor BMS1 on proliferation of neuroblastoma cells

作　　者：郭金鑫 贾安娜 战世佳 张瑶 张璇 郭永丽 常艳 Guo Jinxin;Jia Anna;Zhan Shijia;Zhang Yao;Zhang Xuan;Guo Yongli;Chang Yan(Beijing Key Laboratory for Pediatric Diseases of Otolaryngology,Head and Neck Surgery,Key Laboratory of Major Diseases in Children,Ministry of Education,Beijing Pediatric Research Institute,Beijing Children’s Hospital,Capital Medical University,National Center for Children’s Health,Beijing 100045,China)

机构地区：[1]国家儿童医学中心首都医科大学附属北京儿童医院北京市儿科研究所儿科重大疾病研究教育部重点实验室儿童耳鼻咽喉头颈外科疾病北京市重点实验室,北京100045

出　　处：《首都医科大学学报》2025年第2期296-305,共10页Journal of Capital Medical University

基　　金：国家自然科学基金项目(82402039);北京市自然科学基金项目(7244341,7252046);第三批高层次公共卫生技术人才培养计划项目(领军人才-03-09)。

摘　　要：目的探究核糖体生物合成因子BMS1在神经母细胞瘤(neuroblastoma,NB)细胞增殖中的功能及潜在机制。方法通过R2数据库分析BMS1表达与NB患儿临床特征的相关性;实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测人神经母细胞瘤细胞SK-N-BE(2)、BE(2)-C、IMR-32和正常细胞hTERT RPE-1(人永生化视网膜上皮细胞)、IMR-90(人胚肺成纤维细胞)中BMS1 mRNA水平。利用小分子干扰RNA(small interfering RNA,siRNA)靶向瞬时敲低NB细胞SK-N-BE(2)、BE(2)-C和正常细胞hTERT RPE-1中BMS1 mRNA的表达,RT-qPCR检测BMS1敲低效果及细胞内MYCN mRNA和p53 mRNA水平,并通过结晶紫染色、实时无标记动态细胞分析技术(real time cellular analysis,RTCA)、克隆形成实验、免疫荧光等实验检测细胞增殖活性。结果分析R2数据库中GSE85047(NRC-283)和Westermann-144数据集发现BMS1在MYCN基因扩增的NB样本中表达水平显著高于MYCN基因非扩增的NB样本(P<0.05),且BMS1高表达的NB患儿总体生存率显著降低(P<0.05)。BMS1在NB细胞SK-N-BE(2)、BE(2)-C、IMR-32的mRNA表达水平显著高于正常细胞hTERT RPE-1、IMR-90(P<0.05)。靶向瞬时敲低NB细胞SK-N-BE(2)和BE(2)-C内BMS1导致细胞内MYCN mRNA表达水平下降(P<0.05),细胞的增殖能力和克隆形成能力显著下降(P<0.05),免疫荧光结果显示细胞增殖标志物Ki-67的表达量显著减少(P<0.05)。此外,与对照组相比,SK-N-BE(2)和BE(2)-C细胞中BMS1敲低组(siBMS1-1#和siBMS1-2#)的p53 mRNA水平显著升高(P<0.05)。然而,在正常细胞hTERT RPE-1内敲低BMS1,对细胞增殖无显著影响。结论BMS1在MYCN基因扩增的NB样本中表达上调,且BMS1高表达的NB患儿预后较差;干扰BMS1的表达可转录激活NB细胞内p53从而抑制NB细胞增殖。BMS1能够促进NB的增殖,有望成为NB潜在治疗靶点,尤其是MYCN基因扩增型NB。Objective To elucidate the functional role and underlying mechanisms of the ribosome biogenesis factor BMS1 in neuroblastoma(NB)cellular proliferation.Methods We utilized the R2 genomics analysis and visualization platform to analyze the correlation between BMS1 expression levels and clinical characteristics of NB children.The BMS1 mRNA level in three human neuroblastoma cells SK-N-BE(2),BE(2)-C,IMR-32 and two normal cells hTERT RPE-1,IMR-90 was detected by real-time quantitative polymerase chain reaction(RT-qPCR).Two distinct small interfering RNA(siRNA)sequences were used to target BMS1 mRNA in NB cells SK-N-BE(2)and BE(2)-C,with normal cells hTERT RPE-1 serving as controls.We used RT-qPCR to quantify the mRNA levels of BMS1 and two key neuroblastoma-associated molecules(MYCN and p53).After transfection with siRNA,cellular proliferation was detected by various experimental approaches:crystal violet staining,real-time cell analysis(RTCA),colony-forming unit assay and immunofluorescence.Results By analyzing two independent neuroblastoma clinical cohorts(GSE85047/NRC-283 and Westermann-144 datasets),it was found that the BMS1 mRNA level in MYCN-amplified NB was significantly higher than that in MYCN-non-amplified NB(P<0.05).Furthermore,the overall survival rate of NB children in the BMS1 high-expression group was decreased(P<0.05).Consistent with these clinical observations,the BMS1 mRNA level in NB cells SK-N-BE(2),BE(2)-C and IMR-32 was significantly higher than that in normal cells hTERT RPE-1,IMR-90(P<0.05).The targeted transient knockdown of BMS1 in NB cell lines SK-N-BE(2)and BE(2)-C resulted in decreased intracellular MYCN mRNA expression levels(P<0.05),significantly reduced cell proliferation capacity and colony-forming ability(P<0.05).Immunofluorescence revealed that the expression of Ki-67,a proliferation marker,was decreased(P<0.05).At the molecular level,RT-qPCR showed that the p53 mRNA level was significantly elevated in the BMS1-knockdown groups(siBMS1-1#and siBMS1-2#)compared with the control group(

关 键 词：BMS1 神经母细胞瘤 细胞增殖 P53 MYCN 

分 类 号：R73[医药卫生—肿瘤]