NPM1促进骨肉瘤细胞迁移、增殖、侵袭的体外研究_刘丰平.pdf

海南医学2023年4月第34卷第8期Hainan Med J,Apr.2023,Vol.34,No.8NPM1促进骨肉瘤细胞迁移、增殖、侵袭的体外研究刘丰平，皮闻森，肖运祥，赵红卫三峡大学第一临床医学院脊柱外科，湖北宜昌443000【摘要】目的探讨核仁磷酸蛋白1(NPM1)对骨肉瘤细胞(143B及U2细胞)恶性表型迁移、增殖和侵袭能力的影响。方法采用Oncomine数据库查询NPM1在骨肉瘤中的表达水平；构建重组慢病毒载体，制成过表达NPM1慢病毒(Lv/ShNPM1)、阴性对照慢病毒(Lv/negative)和沉默NPM1慢病毒(Lv/ShNPM1)。分别转染人源骨肉瘤细胞系(143B及U2细胞)，分为Lv/NPM1组、Lv/ShNPM1组、NC(Lv/negative)组。运用 Western blot法检测各组别中NPM1蛋白的表达水平。分别运用CCK8法、Wound healing法、Transwell invasion法检测各组细胞的增殖能力、迁移能力和侵袭能力。结果Oncomine数据库查询结果显示，NPM1在骨肉瘤中呈高表达水平；检测各组中NPM1蛋白表达的结果显示：与NC组比较，Lv/ShNPM1组中NPM1蛋白表达水平降低，差异具有统计学意义(P0.05)；U2-OS 细胞中NC组、Lv/NPM1组和Lv/ShNPM1组细胞迁移率分别为(49.73.3)%、(64.17.4)%、(24.36.6)%，143B细胞分别为(63.54.3)%、(753.7)%、(30.15.9)%，两种细胞Lv/ShNPM1组迁移率明显低于NC组，差异均有统计学意义(P0.05)；U2-OS细胞中NC、Lv/NPM1组、Lv/ShNPM1组侵袭细胞数分别为9826.3、16441、3814.6，143B 细胞分别为10419.1、19132.4、4127.6，两种细胞Lv/ShNPM1组侵袭细胞数明显少于NC组，差异均有统计学意义(P0.05)；CCK8实验结果显示143B和U2-OS细胞Lv/ShNPM1组增殖能力明显低于NC组，差异均有统计学意义(P0.05)。结论NPM1能够体外增强骨肉瘤细胞迁移、增殖和侵袭能力。【关键词】骨肉瘤；核仁磷酸蛋白1；迁移；侵袭；增殖【中图分类号】R738【文献标识码】A【文章编号】10036350（2023）08107204NPM1 promotes the migration,proliferation,and invasion of osteosarcoma cells in vitro.LIU Feng-ping,PIWen-sen,XIAO Yun-xiang,ZHAO Hong-wei.Department of Spinal Surgery,the First Clinical Medical College of ThreeGorges University,Yichang 443000,Hubei,CHINA【Abstract】ObjectiveTo investigate the effect of nucleophosmin 1(NPM1)on the migration,proliferation,and invasion of osteosarcoma cells(143B and U2-OS cells).MethodsBioinformatic prediction was performed to in-vestigate the expression level of NPM1 in osteosarcoma using Oncomine database.Recombinant lentivirus vector wasconstructed to prepare overexpressed NPM1 lentivirus(Lv/ShNPM1),negative control lentivirus(Lv/negative),and si-lent NPM1 lentivirus(Lv/ShNPM1),which were transfected into osteosarcoma cells(143B and U2-OS cells),respec-tively,as Lv/NPM1 group,Lv/ShNPM1 group,NC(Lv/negative)group.Western blot was used to detect the expressionlevel of NPM1 protein in each group.CCK8 method,Wound healing method,and Transwell invasion method were usedto detect the proliferation,migration,and invasion ability of cells in each group.ResultsBioinformatic predictionshowed that NPM1 protein was over-expressed in osteosarcoma cells.Western blot results revealed that the expressionlevels of NPM1 protein was significantly lower in Lv/ShNPM1 group than NC(Lv/negative)group.In U2-OS cells,thecell migration rates in NC(Lv/negative)group,Lv/NPM1 group,and Lv/ShNPM1 group were(49.73.3)%,(64.17.4)%,and(24.36.6)%,respectively;in 143B cells,the cell migration rates were(63.54.3)%,(753.7)%,and(30.15.9)%,respectively;the cell migration rates in Lv/ShNPM1 group were significantly lower than those in NC(Lv/nega-tive)group(P0.05).In U2-OS cells,the number of invasive cells in NC(Lv/negative),Lv/NPM1,and Lv/ShNPM1groups were 9826.3,16441,and 3814.6,respectively;in 143B cells,the number of invasive cells were 10419.1,19132.4,and 4127.6,respectively;the number of invasive cells in Lv/ShNPM1 group were significantly lower than those inNC(Lv/negative)group(P0.05).CCK8 experiment results showed that the proliferation ability of 143B and U2-OS cellsin Lv/ShNPM1 group was significantly lower than that in NC(Lv/negative)group(P0.05).ConclusionNPM1 pro-motes the migration,proliferation,and invasion of osteosarcoma cells in vitro.【Key words】Osteosarcoma;Nucleophosmin 1(NPM1);Migration;Invasion;Proliferation 论著 doi:10.3969/j.issn.1003-6350.2023.08.002基金项目：2020年湖北省宜昌市医疗卫生研究项目资助项目(编号：A20-2-007)。第一作者：刘丰平(1977)，男，副主任医师，研究方向为骨外科临床及基础。通讯作者：皮闻森(1992)，男，研究方向为骨肿瘤转移及其机制，E-mail：。骨肉瘤(osteosarcoma，OS)是最常见的骨与软组织原发性恶性肿瘤，多发生于儿童和青少年。目前，手术治疗、放疗和大剂量化疗是骨肉瘤的主要治疗方法。虽然近年来新辅助化疗药物使得患者生存率有所提高，但是几十年来五年生存率未见提仍未超过60%。其中，OS患者的死亡原因主要是肺部转移，一旦发生5年生存率低于20%1。因此，为了提高患者生存率，更需进一步阐明OS转移的分子机制，明确OS1072Hainan Med J,Apr.2023,Vol.34,No.8海南医学2023年4月第34卷第8期转移防治的有效的策略及靶点。当前诸多研究已经证实NPM1与肿瘤发生、发展及转移密切相关，是一种在多种恶性肿瘤中高表达的癌基因。本研究采用人源骨肉瘤细胞系(143B、U2-OS细胞株)，探讨沉默和过表达NPM1对骨肉瘤细胞恶性表型迁移、增殖和侵袭能力的影响。1材料与方法1.1材料人源骨肉瘤细胞系143B和人源骨肉瘤细胞系U2-OS细胞株购自中国科学院细胞库(上海)，NPM1过表达、沉默及阴性对照慢病毒均购于上海吉凯基因。侵袭试验小室购于北京萌状科技公司。凝 胶制备 试剂 盒(AR018)购 于 Boster 公 司。DMEM培养液、胎牛血清购于美国Gibco公司。蛋白提取试剂盒(BB-3101)购于BestBio公司。NPM1单克隆抗体(兔抗人)购于美国ABcam公司(美国)。HRP标记山羊抗鼠多克隆抗体、-actin 单克隆抗体(鼠抗人)和HRP标记山羊抗兔多克隆抗体均购于中杉金桥公司(北京)。1.2实验方法1.2.1生物信息学预测于Oncomine数据库进行检索，设置类别为骨肉瘤，得到NPM1在骨肉瘤中的表 达 水 平 情 况(https:/ blot 实验检测蛋白表达转染细胞72 h后消化、收集上述各组细胞，提取细胞蛋白，经BCA蛋白定量法测定蛋白含量。SDS-PAGE分离蛋白，于冰上湿转，依照Western blotting 实验手册操作，其中抗兔 NPM1 单克隆抗体比例为 12 000；抗鼠-actin 抗体比例为12 000；辣根过氧化物酶标记的山羊抗小鼠及山羊抗兔IgG比例为 15 000。洗膜后，加入ECL显色液，暗室内曝光。1.2.4CCK-8法检测细胞增殖能力将转染72 h后的U2-OS和143B细胞(3 000/孔)用于试验操作，96孔培养板培养，时间为0 h、24 h和48 h。依照CCK-8法操作手册，每份实验平行做6次。1.2.5Wound healing法检测细胞迁移能力转染72 h后各组细胞，用于试验操作，胰蛋白酶消化后接种于6孔培养板内(细胞密度为5105/mL，500 L/孔，每种细胞5孔)。依照Wound healing法操作手册，每份实验平行做6次。试验结果采用Image J 1.47H软件分析。1.2.6Transwell invasion法检测细胞侵袭能力转染 72 h 后，胰蛋白酶消化各组细胞，用无血清培养基重悬，接种于 Transwell 侵袭小室内(细胞密度为1105个/mL，150 L/室)，依照Transwell invasion法操作手册进行操作。试验结果采用Image J 1.47H软件分析1.3统计学方法应用SPSS13.0 统计软件分析数据，统计作图软件为Raphpad6.0，计量数据以均数标准差(x-s)表示，两组比较采用独立样本t检验，多组间比较采用单因素方差分析。以P0.05为差异有统计学意义。2结果2.1生物信息学结果于Oncomine数据库进行检索显示，NPM1在骨肉瘤中呈高表达，见图1。2.2NPM1转染效率的验证Western blot实验检测Lv/ShNPM1组、NC组和Lv/NPM1组中NPM1蛋白表达情况。结果显示：与NC组比较，Lv/ShNPM1组NPM1蛋白水平明显降低，Lv/NPM1组NPM1蛋白水平增高，差异均有统计学意义(P0.05)，见图2。2.3NPM1增强骨肉瘤细胞的迁移能力U2-OS细胞中NC组、Lv/NPM1组和Lv/ShNPM1组细胞迁移率分别为(49.73.3)%、(64.17.4)%、(24.36.6)%，143B细胞分别为(63.54.3)%、(753.7)%、(30.15.9)%。两图1生物信息学分析结果Figure 1Results of bioinformatics analysis图2Western blot检测NPM1蛋白的表达Figure 2 Detection of NPM1 protein expression by Western blot注：1，NC组；2，Lv/NPM1组；3，Lv/ShNPM1组。Note:1,NC group;2,Lv/NPM1 group;3,Lv/ShNPM1 group.1073海南医学2023年4月第34卷第8期Hainan Med J,Apr.2023,Vol.34,No.8种细胞Lv/ShNPM1组迁移率明显低于NC组，差异均有统计学意义(P0.05)，见图3。2.4NPM1增强骨肉瘤细胞的侵袭能力 U2-OS细胞中NC组、Lv/NPM1组、Lv/ShNPM1组侵袭细胞数分别为9826.3、16441、3814.6，143B细胞分别为10419.1、19132.4、4127.6。两种细胞Lv/ShNPM1组侵袭细胞数明显少于NC组，差异有统计学意义(P0.05)，见图4。2.5NPM1增强骨肉瘤细胞的增殖能力CCK8法检测各组细胞的增殖能力，结果显示 143B 和U2-OS细胞Lv/ShNPM1组增殖能力明显低于NC组，差异有统计学意义(P0.05)，见图5。图3Wound healing 实验检测骨肉瘤细胞的迁徙能力(200)Figure 3The migratory ability of osteosarcoma cells was detected by the Wound healing assay(200)注：1，NC组；2，LV/NPM1组；3，LV/ShNPM1组；与NC组比较，aP0.05。Note:1:NC group;2:Lv/NPM1 group;3:Lv/ShNPM1 group;Compared with NC group,aP0.05.图4Transwell invasion实验检测U2-OS细胞和143B细胞的侵袭能力(200)Figure 4The invasion ability of U2-OS cells and 143B cells was detected by Transwell invasion assay(200)注：1，NC组；2，Lv/NPM1组；3，Lv/ShNPM1组；与NC组比较，aP0.05。Note:1:NC group;2:Lv/NPM1 group;3:Lv/ShNPM1 group;Compared with NC group,aP0.05.3讨论NPM1(nucleophosmin 1)是一种由294个氨基酸组成的具有高活性的核仁蛋白。其具有多种分子生物学功能，包括核糖体蛋白组装，染色质重塑等2。经过磷酸化、乙酰化和泛素化等多种翻译后修饰，NPM1在细胞核和细胞质之间不断穿梭，所处于的亚细胞区域不同，导致行使的功能也不一样3。也有一些研究表明，NPM1在细胞生长、增殖和转化中起着至关重要的作用，可以调节细胞周期进程和中心体复制4。大量研究已经证实在人血液系统恶性肿瘤中检测到突变的NPM1，特别是急性髓性白血病(AML)。NPM1可能是AML的良好预后标志物，甚至是血液图5CCK8实验检测U2-OS细胞和143B细胞的增殖能力Figure 5The proliferation ability of U2-OS cells and 143B cells wasdetected by CCK8 assay1074Hainan Med J,Apr.2023,Vol.34,No.8海南医学2023年4月第34卷第8期系统恶性肿瘤的潜在治疗靶点5-6。与AML相反，NPM1的突变在实体瘤中很少被检测到。但是，越来越多的证据表明，NPM1的在多种实体瘤中表达增高与预后不良有关，包括膀胱尿路上皮癌7、胃癌8。另外，最新的一项纳入了11项研究和997例患者的荟萃分析也表明NPM1的过表达是大多数实体肿瘤预后不良的潜在独立预测因子，包括尤文氏肉瘤、肝细胞癌、胃癌、卵巢浆液性癌、结直肠癌等9。Loubeau等10报道了沉默NPM1能抑制前列腺癌细胞的生长和MAPK信号的传到。最新的研究报道了NPM1可能是前列腺癌免疫诊断和预后的潜在生物标志物，从而弥补PSA缺乏特异性的缺点11。Liu等12证实运用siRNA下调NPM1后能够抑制结直肠癌细胞的恶性表型。本研究中通过生物信息学预测显示NPM1在骨肉瘤中高表达。以此推测NPM1在人骨肉瘤侵袭转移中同样扮演着重要角色。为此，进一步通过体外实验发现沉默NPM1降低了骨肉瘤细胞的侵袭、增殖和迁移能力，而过表达NPM1增强了骨肉瘤细胞的侵袭、增殖和迁移能力。但是NPM1在骨肉瘤中发挥作用的内在分子机制仍然不清楚。NPM1能够调节一些重要的肿瘤抑制因子，如p53和ARF的活性和稳定性13，还可以通过与转录因子NF-B 和 c-Myc 相互作用，从而参与转录激活14-15。Boudra等16报道了mTOR密切参与NPM1基因表达的转录和转录后调节，从而引起NPM1的高表达。另外一项研究证实RNAi沉默NPM1基因能下调前列腺癌细胞中ERK1/2的磷酸化水平17。ERK的磷酸化能够促使NF-从包浆转移至核内18，从而促使MMP-2、MMP-9等与肿瘤转移相关的基因表达上调，促进诸多肿瘤转移，其中包括骨肉瘤19-20。Shandilya等21报道了Aurora-B通过磷酸化NPM1发挥其调节有丝分裂的作用。研究已经证实下调Aurora-B通过NF-信号通路抑制骨肉瘤恶性表型22。总之NPM1在骨肉瘤中发挥作用的内在分子机制仍有需更深入的探索研究。综上所述，本研究证实了NPM1能够在体外促进骨肉瘤细胞的恶性表型。为今后进一步研究NPM1参与骨肉瘤的发生、远处转移及可能机制打下前期基础。参考文献1 Wang Z,Li B,Ren Y,et al.T-cell-based immunotherapy for osteosar-coma:challenges and opportunities J.Front Immunol,2016,7:353.2 Lindstrom MS.NPM1/B23:A multifunctional chaperone in ribosomebiogenesis and chromatin remodeling J.Biochem Res Int,2011,2011:195209.3Handschuh L,Wojciechowski P,Kazmierczak M,et al.NPM1 alter-native transcripts are upregulated in acute myeloid and lymphoblasticleukemia and their expression level affects patient outcome J.JTransl Med,2018,16(1):232.4Okuda M.The role of nucleophosmin in centrosome duplication J.Oncogene,2002,21(40):6170-6174.5Handschuh L,Wojciechowski P,Kazmierczak M,et al.NPM1 alter-native transcripts are upregulated in acute myeloid and lymphoblasticleukemia and their expression level affects patient outcome J.JTransl Med,2018,16(1):232.6 Perry M,Bertoli S,Rocher C,et al.FLT3-TKD mutations associatedwith NPM1 mutations define a favorable-risk group in patients withacute myeloid leukemia J.Clin Lymphoma Myeloma Leuk,2018,18(12):e545-e550.7 Wang H,Yuan G,Zhao B,et al.High expression of B23 is associatedwith tumorigenesis and poor prognosis in bladder urothelial carcino-ma J.Mol Med Rep,2017,15(2):743.8Zhou F,Chen E,You D,et al.Both high expression of nucleophos-min/B23 and CRM1 predicts poorer prognosis in human gastric can-cer J.APMIS,2016,124(12):1046-1053.9 Chen S,He H,Wang Y,et al.Poor prognosis of nucleophosmin over-expression in solid tumors:a meta-analysis J.BMC Cancer,2018,18(1):838.10 Loubeau G,Boudra R,Maquaire S,et al.NPM1 silencing reduces tu-mour growth and MAPK signalling in prostate cancer cells J.PLoSOne,2014,9(5):e96293.11 Dai L,Li J,Xing M,et al.Using serological proteome analysis toidentify serum anti-nucleophosmin 1 autoantibody as a potential bio-marker in European-American and African-American patients withprostate cancer J.Prostate,2016,76(15):1375-1386.12 Liu Y,Zhang F,Zhang XF,et al.Expression of nucleophosmin/NPM1 correlates with migration and invasiveness of colon cancercells J.J Biomed Sci,2012,19(1):53.13 Grisendi S,Mecucci C,Falini B,et al.Nucleophosmin and cancerJ.Nat Rev Cancer,2006,6(7):493-505.14 Li Z,Boone D,Hann SR.Nucleophosmin interacts directly withc-Myc and controls c-Myc-induced hyperproliferation and transfor-mation J.2008,105(48):18794-18799.15 Lin J,Kato M,Nagata K,et al.Efficient DNA binding of NF-kappaBrequires the chaperone-like function of NPM1 J.Nucleic AcidsRes,2017,45(7):3707-3723.16 Boudra R,Lagrafeuille R,Lours-Calet C,et al.mTOR transcriptional-ly and post-transcriptionally regulates Npm1 gene expression to con-tribute to enhanced proliferation in cells with Pten inactivation J.Cell Cycle,2016,15(10):1352-1362.17 Loubeau G,Boudra R,Maquaire S,et al.NPM1 silencing reduces tu-mour growth and MAPK signalling in prostate cancer cells J.PLoSOne,2014,9(5):e96293.18 Han D,Wu G,Chang C,et al.Disulfiram inhibits TGF-beta-inducedepithelial-mesenchymal transition and stem-like features in breastcancer via ERK/NF-kappaB/Snail pathway J.Oncotarget,2015,6(38):40907-40919.19 Chen QJ,Shi Y,Shi JF,et al.Liver X receptors agonist T0901317downregulates matrix metalloproteinase-9 expression in non-small-celllung cancer by repressing nuclear factor-kappaB J.AnticancerDrugs,2017,28(9):952-958.20 Lin YW,Lee LM,Lee WJ,et al.Melatonin inhibits MMP-9 transacti-vationandrenalcellcarcinomametastasisbysuppressingAkt-MAPKs pathway and NF-kappaB DNA-binding activity J.J Pi-neal Res,2016,60(3):277-290.21 Shandilya J,Senapati P,Dhanasekaran K,et al.Phosphorylation ofmultifunctional nucleolar protein nucleophosmin(NPM1)by aurorakinase B is critical for mitotic progression J.FEBS Lett,2014,588(14):2198-2205.22 Zhu LB,Jiang J,Zhu XP,et al.Knockdown of Aurora-B inhibits os-teosarcoma cell invasion and migration via modulating PI3K/Akt/NF-kappaB signaling pathway J.Int J Clin Exp Pathol,2014,7(7):3984-3991.(收稿日期：2022-06-19)1075