NOX2通过干预ROS介导的PI3K/AKT信号通路对多发性骨髓瘤细胞能量代谢重编程的作用  

Effect of NOX2 on energy metabolism reprogramming of multiple myeloma cells by interfering with ROS-mediated PI3K/AKT signal pathway

作　　者：卓理想 梁冰[2] 张梦琼 陈夏 ZHUO Lixiang;LIANG Bing;ZHANG Mengqiong;CHEN Xia(Department of Hematology,The First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,Anhui,China;Department of Gerontology,The First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,Anhui,China;Department of Hematology,Suzhou first people's Hospital,Suzhou 234000,Anhui,China)

机构地区：[1]蚌埠医学院第一附属医院血液内科,安徽蚌埠233004 [2]蚌埠医学院第一附属医院老年病科,安徽蚌埠233004 [3]宿州市第一人民医院血液内科,安徽宿州234000

出　　处：《西部医学》2024年第10期1427-1435,共9页Medical Journal of West China

摘　　要：目的探讨烟酰胺腺嘌呤二核苷酸磷酸氧化酶2(NOX2)通过干预活性氧(ROS)介导的磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号通路对多发性骨髓瘤(MM)细胞能量代谢重编程的作用。方法收集2019年4月-2022年4月蚌埠医学院第一附属医院收治的50例MM患者骨髓血为MM组,同时收集29例健康正常人骨髓血为对照组,qRT-PCR检测骨髓血NOX2、PI3K相对表达、MM细胞系U266、LP-1的NOX2相对表达水平,分析NOX2与PI3K表达水平、NOX2表达水平与临床病理特征的相关性。WB检测U266、LP-1细胞PI3K/AKT信号通路相关分子表达水平,2′,7′-二氯二氢荧光素二乙酯(DCFH-DA)荧光探针检测细胞ROS水平,2-N(7-硝基苯-2-乙二酸,3-4羟氨基)-2-脱氧葡萄糖(2-NBDG)检测细胞葡萄糖摄取能力,生化实验检测细胞乳酸分泌水平,Seahorse检测细胞代谢情况,通过CCK-8、EdU法、划痕、Transwell实验与体内异种移植实验检测细胞增殖、迁移、侵袭及成瘤能力,免疫组化检测移植瘤NOX2、PI3K/AKT信号通路及糖酵解相关分子表达情况。结果NOX2表达水平在MM患者骨髓血、MM细胞系中均升高。相关性分析结果显示,MM患者骨髓血NOX2与PI3K表达水平呈正相关,ISS分期、β2-微球蛋白、Durie-Salmon分期与NOX2表达水平相关(均P<0.05)。与NC组、sh-NC组相比,NOX2组p-PI3K、p-AKT表达水平升高,sh-NOX2组p-PI3K、p-AKT表达水平降低(均P<0.05)。与NC组相比,NOX2组ROS水平、2-NBDG荧光强度、细胞乳酸分泌水平、细胞外酸化率、糖酵解能力、增殖、迁移、侵袭能力升高,移植瘤体积、质量增大,肿瘤乳酸脱氢酶A(LDHA)、3-磷酸肌醇依赖性蛋白激酶1(PDK1)、p-PI3K、p-AKT表达水平升高(均P<0.05)。结论NOX2在MM患者骨髓血与细胞系中高表达,可能通过促进ROS生成激活PI3K/AKT信号通路,促进MM细胞能量代谢重编程与恶性生物学行为,进而推动MM发展。Objective To investigate the effect of nicotinamide adenine dinucleotide phosphate oxidase 2(NOX2)on energy metabolism reprogramming of multiple myeloma(MM)cells by interfering with phosphatidylinositol 3 kinase/protein kinase B(PI3K/AKT)signal pathway mediated by reactive oxygen species(ROS).Methods The bone marrow blood of 50 patients with MM and 29 healthy controls were collected from April 2019 to April 2022.The relative expressions of NOX2,PI3K in bone marrow blood and the level of NOX2 in MM cell line U266 and LP-1 were detected by qRT-PCR.To analyze the correlation between the expression levels of NOX2 and PI3K,NOX2 and clinicopathological features.Detection of PI3K/AKT signal pathway related molecules expression in U266 and LP-1 cells by WB.Detection of ROS level in cells by 2',7'-dichlorofluorescin diacetate(DCFH-DA)fluorescence probe.2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose(2-NBDG)was used to detect the glucose uptake of cells.Detection of lactic acid secretion was applied by biochemical experiment.Detection of cell metabolism was applied by Seahorse.Cell proliferation,migration,invasion and tumorigenesis were detected by CCK-8,EdU,scratch,Transwell and xenotransplantation in vivo.Immunohistochemical detection of NOX2,PI3K/AKT signal pathway and glycolysis related molecule expression in transplanted tumor.Results The expression of NOX2 increased in bone marrow blood of MM patients and MM cell line.The results of correlation analysis showed that there was a positive correlation between the expression level of NOX2 and PI3K in bone marrow blood of MM patients,and the expression level of NOX2 was correlated with ISS stage,β2-microglobulin and Durie-Salmon stage.Compared with NC group and sh-NC group,the expression of p-PI3K and p-AKT in U266 and LP-1 cells increased in NOX2 group(P<0.05),while p-PI3K and p-AKT expression decreased in sh-NOX2 group(P<0.05).Compared with NC group,ROS level,2-NBDG fluorescence intensity,lactic acid secretion,extracellular acidification rate,glycolysis ab

关 键 词：烟酰胺腺嘌呤二核苷酸磷酸氧化酶2 活性氧 磷脂酰肌醇3激酶 蛋白激酶B 多发性骨髓瘤 

分 类 号：R733.5[医药卫生—肿瘤]