E2F转录因子1抑制神经母细胞瘤细胞衰老的作用及其机制  

Roles of E2F transcription factor 1 in regulating cellular senescence of neuroblastoma and its mechanisms

作　　者：杜昕怡 李聃 童强松[1] Du Xinyi;Li Dan;Tong Qiangsong(Department of Pediatric Surgery,Union Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430022,China)

机构地区：[1]华中科技大学同济医学院附属协和医院小儿外科,武汉430022

出　　处：《中华实验外科杂志》2024年第2期255-258,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(82072801、82173316、82293663)。

摘　　要：目的探讨E2F转录因子1(E2F1)对神经母细胞瘤(NB)细胞衰老的调控作用及机制。方法人神经母细胞瘤细胞株SK-N-BE (2) (CRL-2271)购自美国典型培养物保藏中心, 并将NB细胞株分为空载体(Mock)对照组、E2F1过表达组、随机干扰(sh-Scb)组、E2F1短发卡RNA (sh-E2F1)干扰组, 筛选稳定亚克隆细胞株;采用实时定量反转录聚合酶链反应(RT-qPCR)、蛋白质印迹法(Western blot)检测NB细胞中E2F1表达变化;通过Kaplan-Meier Plot、ChEA3等数据库分析, 寻找NB细胞衰老相关靶基因;衰老相关β-半乳糖苷酶(SA-β-Gal)染色12 h后, 检测β-半乳糖苷酶活性, 计算衰老细胞阳性率;5-乙炔基-2’-脱氧尿苷(EdU)掺入细胞2 h后, 观测细胞增殖活性;软琼脂三维培养3周, 观测细胞克隆形成能力;基质胶侵袭24 h, 测细胞侵袭活性;组间比较采用t检验。结果过表达E2F1组的E2F1转录水平高于Mock组(3.24±0.12比1.00±0.04, t=32.06, P<0.05), 且E2F1蛋白水平也高于Mock组;而E2F1敲低组中E2F1转录本水平低于sh-Scb组(0.67±0.04比1.00±0.03, t=14.24, P<0.05;0.67±0.08比1.00±0.03, t=13.80, P<0.05), 蛋白水平也低于sh-Scb组。E2F1过表达组胰岛素样生长因子结合蛋白2(IGFBP2)的转录本和蛋白水平高于Mock组(2.17±0.15比1.02±0.00, t=13.00, P<0.05)且E2F1过表达组聚合酶γ基因(POLG)转录本和蛋白水平高于Mock组(2.38±0.11比1.02±0.01, t=15.20, P<0.05)。衰老细胞阳性率下降(6.66±1.52比23.33±2.51, t=9.80, P<0.05), 细胞增殖速率增高(51.66±1.52比23.00±4.00, t=11.60, P<0.05), 克隆形成数增加(334.66±6.11比157.66±4.16, t=41.46, P<0.05)且侵袭细胞数增加(318.33±8.02比126.33±5.50, t=34.18, P<0.05)。相反, E2F1干扰组中IGFBP2转录水平低于随机干扰组(0.38±0.06比1.02±0.01, t=17.05, P<0.05;0.33±0.05比1.02±0.01, t=19.76, P<0.05), IGFBP2蛋白水平也低于随机干扰组。POLG转录水平降低于随机干扰组(0.50±0.02比1.02±0.01, t=30.35, P<0.05;0.35±0.05比1.02±0.01, tObjective To investigate regulatory effect and mechanisms of E2F transcription factor 1(E2F1)on cellular senescence in neuroblastoma(NB).Methods The human neuroblastoma cell line SK-N-BE(2)(CRL-2271)was purchased from the American Type Culture Collection(Rockville,MD).NB cells were divided into empty vector(mock),E2F1 over-expression,scramble short hairpin RNA(shRNA),sh-E2F1#1,and sh-E2F1#2 groups,while stable cells were screened.The E2F1 expression was detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)and Western blotting.Kaplan-Meier Plot,ChEA3 and other databases were used to search possible target genes.Senescence-associatedβ-galactosidase(SA-β-Gal)staining was performed for 12 h to detectβ-galactosidase-positive senescent cells.5-ethynyl-2’-deoxyuridine(EdU)incorporation for 2 h was applied for assessing cellular proliferation.Incubation within soft agar for 3 weeks was undertaken to assess cell growth.Matrigel transwell assay for 24 h was applied for observing cellular invasion capability.Student’s t-test was applied for statistical analysis.Results In subclonal lines over-expressing E2F1,the transcript and protein levels increased compared with mock group(3.24±0.12 vs.1.00±0.04,t=32.06,P<0.05).Meanwhile,its transcript levels were reduced in E2F1 silencing groups as compared with sh-Scb group(0.32±0.08 vs.1.00±0.03,t=14.24,P<0.05;0.33±0.08 vs.1.00±0.03,t=13.80,P<0.05).Similarly,its protein levels were reduced.As compared with the mock group,insulin like growth factor binding protein 2(IGFBP2)transcript and protein levels increased in E2F1 over-expression group(2.17±0.15 vs.1.02±0.00,t=13.00,P<0.05).DNA polymerase gamma catalytic subunit(POLG)transcript and protein levels increased in E2F1 over-expression group(2.38±0.11 vs.1.02±0.01,t=15.20,P<0.05).Senescent cell positivity decreased(6.66±1.52 vs.23.33±2.51,t=9.80,P<0.05),and proliferation rate of tumor cells increased(51.66±1.52 vs.23.00±4.00,t=11.60,P<0.05).The number of clone formation and inf

关 键 词：神经母细胞瘤 E2F转录因子1 细胞衰老 

分 类 号：R739.4[医药卫生—肿瘤]