沉默NUPR1降低人多发性骨髓瘤U266细胞自噬并促进其迁移和侵袭  

Silencing NUPR1 down-regulates autophagy and promotes migration and invasion in human multiple myeloma U266 cells

作　　者：李星欣 黎安茂 曾沉思 陈建斌[1] LI Xingxin;LI Anmao;ZENG Chensi;CHEN Jianbin(Departments of Hematology,the First Affiliated Hospital of Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学附属第一医院血液内科

出　　处：《第三军医大学学报》2019年第23期2322-2327,共6页Journal of Third Military Medical University

摘　　要：目的探讨沉默核蛋白1(nuclear protein 1,NUPR1)对人多发性骨髓瘤细胞自噬、迁移和侵袭的影响及其机制。方法用含有NUPR1-shRNA慢病毒载体的病毒感染骨髓瘤U266细胞,通过流式细胞术检测转染效率,实验分为阴性对照组(转染无关序列shRNA慢病毒)和敲低组(转染NUPR1-shRNA慢病毒)。qRT-PCR检测NUPR1mRNA表达,Western blot检测NUPR1、自噬相关蛋白(LC3Ⅱ/LC3Ⅰ、Beclin1、ATG5和P62)、迁移和侵袭相关蛋白(MMP9、CXCR4)以及通路蛋白(p-AKT/T-AKT和p-mTOR/T-mTOR)的表达;透射电子显微镜(TEM)观察自噬小体;Transwell检测细胞迁移和侵袭能力。结果与阴性对照组比较,敲低组NUPR1 mRNA和蛋白表达显著下调(P<0.05),自噬相关蛋白ATG5、Beclin1和LC3Ⅱ/LC3Ⅰ的表达明显降低(P<0.05),而MMP9、CXCR4、P62、p-AKT/T-AKT和p-mTOR/T-mTOR蛋白表达明显增高(P<0.05);敲低组自噬小体明显减少,细胞迁移和侵袭能力增强,雷帕霉素可阻断该促进作用。结论沉默NUPR1可能通过降低自噬水平促进U266细胞迁移和侵袭,并可能与AKT/mTOR通路激活有关。ObjectiveTo explore the effect of silencing nuclear protein 1(NUPR1)on autophagy,migration and invasion in human multiple myeloma(MM)U266 cells and its possible mechanism.MethodsNUPR1-shRNA was transfected into MM U266 cells,and the transfection rate was detected by flow cytometry.Then the cells were divided into negative control cells(transfected with unrelated sequence RNA lentiviral vector)and NUPR1 knockdown cells(transfected with NUPR1-shRNA lentiviral vector).The interference effect of NUPR1 was detected by qRT-PCR,and the expression levels of autophagy related proteins(Beclin1,ATG5,LC3Ⅱ/LC3Ⅰ,P62),migration and invasion associated proteins(MMP9,CXCR4)and pathway proteins(p-AKT/T-AKT,p-mTOR/T-mTOR)were measured by Western blotting.Transmission electron microscopy(TEM)was used to observe autophagosomes.Transwell test was employed to detect cell migration and invasion.ResultsThe expression of NUPR1 at mRNA and protein level was significantly decreased in NUPR1 knockdown cells(P<0.05).The protein levels of autophagy related proteins,ATG5,Beclin1 and LC3Ⅱ/LC3Ⅰwere significantly lower,while those of f MMP9,CXCR4,P62,p-AKT/T-AKT and p-mTOR/T-mTOR were increased obviously in the NUPR1 knockdown cells than the negative control cells(P<0.05).There were significantly reduced autophagosomes in NUPR1 knockdown cells.While,migratory and invasive abilities were enhanced in NUPR1 knock down group,and rapamycin reduced the promoting effects.ConclusionSilencing NUPR1 may promotes migration and invasion by down-regulating autophagy in U266 cells,which may be related to activation of the AKT/mTOR pathway.

关 键 词：核蛋白1 多发性骨髓瘤 自噬 迁移 侵袭 

分 类 号：R73-37[医药卫生—肿瘤] R730.23[医药卫生—临床医学]