沙门菌致病岛1蛋白促进神经母细胞瘤Warburg效应的作用及分子机制  

Salmonella pathogenicity island 1 promotes neuroblastoma progression through enhancing Warburg effect

作　　者：金石开 袁博玲 李聃 童强松[1] Jin Shikai;Yuan Boling;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》2022年第1期81-84,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81874085、81802925、81903011、81903008、82072801、82173316)。

摘　　要：目的探讨沙门菌致病岛1(SPI1)蛋白在神经母细胞瘤中调控有氧糖酵解(即Warburg效应)的作用及分子机制。方法运用公用数据库,解析神经母细胞瘤中调控Warburg效应的候选关键转录因子SPI1;经嘌呤霉素筛选,获得稳定过表达/敲低SPI1的SK-N-BE(2)细胞株,分为空载体对照(Mock)组、SPI1过表达(SPI1)组、随机干扰(sh-Scb)组、SPI1干扰(sh-SPI1)组;实时荧光定量聚合酶链反应(PCR)、染色质免疫共沉淀、双荧光素酶报告基因及蛋白质印迹法(Western blot)检测靶基因启动子活性和表达水平;分光光度计法和糖酵解压力试验检测有氧糖酵解水平;软琼脂克隆形成和基质胶侵袭检测瘤细胞的增殖和侵袭活性。组间比较采用t检验。结果SPI1组己糖激酶2(HK2)表达高于Mock组(3.63±0.53比1.00±0.05,t=17.462,P<0.05),磷酸甘油酸激酶1(PGK1)表达高于Mock组(3.28±0.39比1.00±0.03,t=17.651,P<0.05),细胞外酸化率(ECAR)高于sh-Scb组[(32.17±3.15)mpH/min比(9.82±1.49)mpH/min,t=10.352,P<0.05],克隆形成数高于Mock组[(163.30±7.59)个比(48.02±4.85)个,t=17.840,P<0.05],侵袭细胞数高于Mock组[(184.70±10.58)个比(88.33±5.56)个,t=8.214,P<0.05]。sh-SPI1组HK2表达低于sh-Scb组(0.33±0.02比1.00±0.04,t=14.370,P<0.05),PGK1表达低于sh-Scb组(0.34±0.02比1.00±0.03,t=19.192,P<0.05),ECAR值低于sh-Scb组(3.07±0.48比10.05±1.37,t=7.580,P<0.05),克隆形成数低于sh-Scb组[(21.33±3.96)个比(44.67±5.69)个,t=5.916,P<0.05],侵袭细胞数低于sh-Scb组[(40.67±3.48)个比(88.00±6.89)个,t=4.994,P<0.05],差异均有统计学意义。结论转录因子SPI1通过增强HK2和PGK1表达,促进神经母细胞瘤的Warburg效应和肿瘤进展。Objective To explore the molecular mechanism of salmonella pathogenicity island 1(SPI1)regulating aerobic glycolysis(the Warburg effect)in neuroblastoma(NB).Methods Through comprehensive analysis of public databases,SPI1 was found to be a key transcription factor regulating the Warburg effect in NB.The SK-N-BE(2)cell line stably over-expressing or silencing SPI1 was established after puromycin selection.NB cell line was divided into mock,SPI1,sh-Scb,and sh-SPI1 groups.Chromatin immunoprecipitation,real-time quantitative PCR,dual luciferase reporter,and Western blotting assays were applied to detect the promoter activity and expression levels of target genes.The regulation of aerobic glycolysis was investigated by spectrophotometry and glycolysis stress test.Soft agar and matrigel invasion assays were used to detect the proliferative and invasive abilities of NB cell line.Student’s t-test was applied for statistical analysis.Results The expression levels of hexokinase 2(HK2)(3.63±0.53 vs.1.00±0.05,t=17.462,P<0.05)and phosphoglycerate kinase 1(PGK1)(3.28±0.39 vs.1.00±0.03,t=17.651,P<0.05)in SPI1 group were higher than those in mock group.The extracellular acidification rate(ECAR)in SPI1 group was higher than that in mock group[(32.17±3.15)mpH/min vs.(9.82±1.49)mpH/min,t=10.352,P<0.05].The number of cell colony formation in SPI1 group was greater than that in mock group(163.30±7.59 vs.48.02±4.85,t=17.840,P<0.05),and the number of invasive cells was greater than that in mock group(184.70±10.58 vs.88.33±5.56,t=8.214,P<0.05).In contrast,the expression levels of HK2(0.33±0.02 vs.1.00±0.04,t=14.370,P<0.05)and PGK1(0.34±0.02 vs.1.00±0.03,t=19.192,P<0.05)in sh-SPI1 group were lower than those in sh-Scb group.The ECAR in sh-SPI1 group was lower than that in sh-Scb group[(3.07±0.48)mpH/min vs.(10.05±1.37)mpH/min,t=7.580,P<0.05].The number of cell colony formation in sh-SPI1 group was less than that in sh-Scb group(21.33±3.96 vs.44.67±5.69,t=5.916,P<0.05),and the number of invasive cells was less than that

关 键 词：神经母细胞瘤 基因转录 沙门菌致病岛1 

分 类 号：R730.2[医药卫生—肿瘤]