摘要
目的 研究蛋白磷酸酶1调节亚基14B(PPP1R14B)对神经母细胞瘤(NB)细胞有丝分裂进程的调控作用,为其治疗提供潜在新靶标。方法 在NB细胞SK-N-BE(2)-M17中转染2条不同序列小干扰RNA(siRNA)(PPP1R14B siRNA#1和#2)敲低PPP1R14B,实时定量PCR和Western印迹法检测PPP1R14B的敲低效果。在SK-N-BE(2)-M17 GFP-H2B细胞中转染PPP1R14B siRNA#1和#2,Time-lapse活细胞成像技术动态监测敲低PPP1R14B对该细胞有丝分裂进程的影响。采用酶切克隆法构建靶向PPP1R14B的2条不同序列短发夹RNA(shRNA)(sh PPP1R14B#1或#2)慢病毒质粒,并感染SK-N-BE(2)-M17细胞建立PPP1R14B稳定敲低细胞,Western印迹法检测其敲低效果,克隆形成实验观察敲低PPP1R14B对SK-N-BE(2)-M17细胞生长的影响。基于GEO数据库的临床数据,按NB不同临床分期分组,分析各组样本中PPP1R14B mRNA表达水平及其与预后的相关性。结果 与转染对照siRNA相比,转染PPP1R14B siRNA#1和#2均可有效敲低SK-N-BE(2)-M17细胞中PPP1R14B mRNA和蛋白表达(P<0.01)。Timelapse活细胞成像结果显示,与转染对照siRNA相比,转染PPP1R14B siRNA#1和#2组SK-N-BE(2)-M17GFP-H2B细胞有丝分裂期时间延长(P<0.05),且主要是有丝分裂前中期时间显著延长(P<0.01)。Western印迹法结果显示,与转染对照shRNA相比,转染sh PPP1R14B#1和#2均可有效敲低SK-N-BE(2)-M17细胞中PPP1R14B蛋白表达(P<0.01);克隆形成实验结果表明,转染sh PPP1R14B#1或#2稳定敲低组细胞克隆形成数亦明显降低(P<0.01)。GEO数据库分析显示,PPP1R14B mRNA表达水平与NB恶性程度呈正相关,与患者预后呈负相关。结论 敲低PPP1R14B导致SK-N-BE(2)-M17细胞有丝分裂期阻滞和生长减慢,PPP1R14B在NB发生发展中发挥重要作用。
OBJECTIVE To study the regulatory effect of protein phosphatase 1 regulatory subunit 14B(PPP1R14B)on mitotic progression of neuroblastoma(NB)cells so as to recommend a potential new target for neuroblastoma therapy.METHODS Two small interfering RNAs(siRNAs)with different sequences(PPP1R14B siRNA#1 and#2)were transfected into NB cells SK-N-BE(2)-M17 to knock down PPP1R14B.The knockdown effect of PPP1R14B was detected by real-time quantitative PCR and Western blotting.The time-lapse living cell imaging technique was used to dynamically monitor the effect of knocking down PPP1R14B on mitotic progression of SK-N-BE(2)-M17 GFP-H2B cells.Two short hairpin RNA(shRNA)lentiviral plasmids with different sequences(shPPP1R14B#1 and#2)targeting PPP1R14B were constructed using the enzyme digestion cloning method.SK-N-BE(2)-M17 cells were infected to establish PPP1R14B stable knockdown cells,and the knockdown effect was detected by Western blotting.Clone formation experiments were conducted to observe the effect of knockdown of PPP1R14B on the growth of SK-N-BE(2)-M17 cells.Based on the clinical data from the GEO database and different clinical stages of NB,the mRNA expression level of PPP1R14B in each group and its correlation with prognosis were analyzed.RESULTS Compared with control siRNA transfection,both groups transfected with PPP1R14B siRNA#1 and#2 could effectively reduce the expression of PPP1R14B in SK-N-BE(2)-M17 cells(P<0.01).Time-lapse living cell imaging assay showed that the mitotic periods of SK-N-BE(2)-M17 GFP-H2B cells transfected with PPP1R14B siRNA#1 and#2 were prolonged(P<0.05),particularly the time of prometaphase(P<0.01).Western blotting verified that shPPP1R14B#1 and#2 targeting PPP1R14B could effectively reduce the expression of PPP1R14B in SK-N-BE(2)-M17 cells compared with control shRNA(P<0.01).Clone formation assay showed that the number of cell clones in the PPP1R14B stable knockdown groups by shPPP1R14B#1 and#2 respectively was significantly decreased(P<0.01).GEO database analysis showed that the mRNA expression level of PPP1R14B was positively correlated with the malignant degree of NB and negatively with the prognosis of patients.CONCLUSION Knockdown of PPP1R14B leads to mitotic arrest and growth retardation of NB cells,and PPP1R14B plays an important role in the occurrence and development of NB.
作者
简肖肖
张万鹏
常艳
张学敏
李慧艳
胡怀斌
JIAN Xiaoxiao;ZHANG Wanpeng;CHANG Yan;ZHANG Xuemin;LI Huiyan;HU Huaibin(National Center of Biomedical Analysis,Beijing 100850,China;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)
出处
《中国药理学与毒理学杂志》
CAS
北大核心
2023年第9期688-694,共7页
Chinese Journal of Pharmacology and Toxicology
基金
北京市自然科学基金(7212038)。
