电子转移黄素蛋白A对肝癌细胞SMMC-7721恶性生长的影响  

Effect of electron transfer flavoprotein A on oncogenic growth of SMMC-7721 cancer cells

作　　者：刘根玉 赵敏 郭璐 张纪岩 LIU Gen-yu;ZHAO Min;GUO Lu;ZHANG Ji-yan(Institute of Military Cognition and Brain Sciences,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]军事科学院军事医学研究院军事认知与脑科学研究所,北京100850

出　　处：《中国药理学与毒理学杂志》2021年第2期96-101,共6页Chinese Journal of Pharmacology and Toxicology

基　　金：国家杰出青年科学基金(81625010);国家自然科学基金(81601786)。

摘　　要：目的探讨线粒体β氧化关键蛋白电子转移黄素蛋白A(ETFA)对肝癌细胞SMMC-7721恶性生长的影响及潜在的调控机制。方法将携带对照短发夹RNA(shRNA)或人ETFA shRNA 1#和2#的pGPU6/Hygro载体转染SMMC-7721细胞,分别为对照组、ETFA-1#组和ETFA-2#组。通过潮霉素筛选得到稳定敲低ETFA的SMMC-7721细胞克隆,并用Western印迹法检测细胞中ETFA蛋白水平,对稳定敲低细胞进一步鉴定。将筛选得到的稳定细胞用于以下实验。将细胞与0.3%琼脂混匀,均匀铺于6孔板(每孔1×10^(3)细胞),第10天观察集落形成并计数。每只裸小鼠皮下注射2×10^(6)细胞进行皮下成瘤实验,于第14天开始每2 d测量1次瘤体体积,第28天测瘤重。将细胞接种于6孔板,用尼罗红染色法于激光共聚焦显微镜下观察细胞中脂质堆积。将细胞固定后,使用扫描电镜观察细胞超微结构的变化。结果Western印迹法结果显示,ETFA-1#和ETFA-2#组SMMC-7721细胞ETFA的表达水平明显低于对照组,表明稳定敲低ETFA的SMMC-7721细胞构建成功。集落形成实验结果显示,在细胞接种第10天,对照组细胞平均克隆数为99±7,ETFA-1#组为55±5(P<0.01),ETFA-2#组为24±12(P<0.01)。皮下成瘤实验结果显示,在裸小鼠皮下接种细胞28 d后,对照组瘤块平均体积(mm^(3))为1220±474,ETFA-1#组为671±246(P<0.01),ETFA-2#组为534±217(P<0.01);对照组瘤块平均质量(g)为0.65±0.21,ETFA-1#组为0.39±0.13(P<0.01),ETFA-2#组为0.40±0.15(P<0.01)。尼罗红染色结果显示,敲低ETFA的SMMC-7721细胞中可见大量红色斑点,而对照组未见此现象。在透射电镜下,敲低ETFA的SMMC-7721细胞中可见数量较多、体积较大的脂滴,而对照组未见明显脂滴。结论敲低ETFA可抑制SMMC-7721细胞体外非锚定生长能力和体内成瘤能力,其机制可能与脂质代谢障碍有关。OBJECTIVE To investigate the effect of electron transfer flavoprotein A(ETFA)knockdown on oncogenic growth of hepatoma cells and the potential regulatory mechanism.METHODS The pGPU6/Hygro vectors carrying control short hairpin RNA(shRNA)or human ETFA shRNA-1#and 2#were transfected into SMMC-7721 cells which were identified as control,ETFA-1#and ETFA-2#groups,respectively.After hygromycin screening,SMMC-7721 cells with ETFA knockdown were identified by Western blotting to detect the level of ETFA protein.The selected stable clones were seeded into 6-well plates at a density of 1×10^(3) cells per well before the colonies were photoed and counted on the 10th day of inoculation.The selected stable clones were injected subcutaneously into the nude mice at a density of 2×10^(6) cel s per mouse.Tumors were measured every two days from the 14th day,and tumors were removed,photoed and weighed on the 28th day.The selected stable clone cells were seeded on the 6-well plates,and lipid drops in the cells were observed after Nile red staining.The selected stable clones were fixed and the changes of cell microstructure were observed under a scanning electron microscope.RESULTS Western blotting assay showed that the level of ETFA in ETFA-1#and ETFA-2#groups was significantly lower than that in the control group,indicating that the stable clones of knockdown ETFA were constructed in SMMC-7721 cells.Colony formation assay showed that the average number of clones was 99.0±6.6 in the control group,55.3±5.0 in the ETFA-1#group(P<0.01)and 24.3±11.7 in the ETFA-2#group(P<0.01)after ten days of inoculation.Subcutaneous tumor formation essay showed that the average volume(mm3)of tumors was 1220±474 in the control group,671±246 in the ETFA-1#group(P<0.01)and 534±217 in the ETFA-2#group(P<0.01),while the average mass(g)of tumors was 0.65±0.21 in the control group,0.39±0.13 in the ETFA-1#group(P<0.01),and 0.40±0.15 in the ETFA-2#group(P<0.01)after 28 d of inoculation.In Nile red staining assay,a large number of red spots were obs

关 键 词：电子转移黄素蛋白A 脂肪酸 β氧化 肝细胞癌 

分 类 号：R735.7[医药卫生—肿瘤]