eIF3g差异表达的乳癌细胞模型的建立

Establishment of Breast Cancer Cell Models with Inducible Differential eIF3g Expressions

真核翻译起始因子3的亚单位eIF3g在一些多药耐药肿瘤细胞中表达上调。背景相近而eIF3g表达有明显差异的肿瘤细胞模型的建立对阐明其作用及机制有重要意义。该研究利用四环素调控的Tet-On Advanced诱导表达系统,分别构建了可诱导过表达外源性eIF3g和表达eIF3g人工microRNA的载体,并包装为相应的慢病毒,将慢病毒分别感染乳腺癌细胞Bcap37,经400μg/mLG418和0.4μg/mL Puromycin筛选后,分别获得稳定转染的乳癌细胞克隆Bcap37/Tet-On-eIF3g和Bcap37/Tet-On-eIF3gmiR。将这些细胞克隆分别在1μg/mL DOX的作用下诱导培养72 h,用West-ern blot检测eIF3g的表达。结果显示,Bcap37/Tet-On-eIF3g中eIF3g表达明显增加,Bcap37/Tet-On-eIF3gmiR中eIF3g表达抑制明显。该工作成功建立了可诱导外源性eIF3g过表达和抑制内源性eIF3g表达的乳腺癌细胞模型,为进一步的研究打下了基础。

EIF3g, one of the subunits of eukaryotic translation initiation factor 3 complex, is over ex- pressed in some multidrug resistant cancer cells. Establishment of cancer cell models with similar background but significantly different expression patterns of eIF3g is important for elucidating its roles and mechanisms. The tetracycline-inducible expression system Tet-On Advanced was used in this work. Vectors with cDNA of eIF3g full coding region and artificial microRNA targeting eIF3g were constructed and packed into recombinant lentiviruses and used to infect human breast cancer cell Bcap37. Stably-transfected clones were obtained after co-selection by 400 gg/mL G418 and 0.4 μg/mL puromycin, namely Bcap37/Tet-On-elF3g and Bcap37/Tet-On-eIF3gmiR, respectively. Western blot was used to examine the expression of eIF3g in these clones before and after induction with doxycycline （DOX）. The results showed that DOX treatment could significantly up-regulate the expression of eIF3g in Bcap37/Tet-On-eIF3g cells and down-regulate the expression of eIF3g in Bcap37/Tet-On-eIF3gmiR cells. Breast cancer cell models with inducible differential eIF3g expression were therefore established, and this provides the ba- sis for further investigation on eIF3g.