电子转移黄素蛋白A对肝癌细胞SMMC-7721恶性生长的影响

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1×10³细胞),第10天观察集落形成并计数。每只裸小鼠皮下注射2×10⁶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(mm³)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。

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

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 1×10³ 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 2×10⁶ 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 observed in the knockdown clones,but there was no lipid droplet in the control group.Under the electron microscope,a large number of lipid droplets were observed in the knockdown clones,but no significant lipid droplet was found in the control group.CONCLUSION ETFA knockdown can inhibit the oncogenic growth of SMMC-7721 cells.The mechanism may be related to lipid metabolism disorder.