转录因子Twist基因慢病毒质粒构建及其促进乳腺肿瘤上皮细胞MCF-7上皮间质转化

Construction of lentivirus expression vector containing transcription factor Twist gene and its effect on promoting epithelial to mesenchymal transition of breast cancer cells MCF-7

目的 :构建转录因子Twist基因的慢病毒质粒,其高表达促进乳腺肿瘤上皮细胞MCF-7上皮间质转化(epithelial to mesenchymal transition,EMT)。方法 :pcDNA3/myc-tagged-Twist质粒扩增并亚克隆至载体pLVX-puro中构建成pLVX-puromyc-tagged-Twist慢病毒质粒。将其转染人胚肾细胞HEK293T,收取病毒上清感染乳腺癌MCF-7细胞。免疫荧光法和蛋白质印迹法(Western blot)检测转录因子Twist蛋白、上皮标志蛋白E-钙黏蛋白(E-cadherin)、间质标志蛋白波形蛋白(Vimentin)的表达。Transwell法检测细胞侵袭能力。结果:pLVX-puro-myc-tagged-Twist慢病毒质粒酶切及测序结果完全正确;相对于对照细胞(MCF-7-Vector细胞),转染Twist基因的MCF-7细胞(MCF-7-Twist细胞)Twist蛋白、间质标志蛋白Vimentin表达水平明显升高,上皮标志蛋白E-cadherin表达水平明显降低(P<0.05),侵袭能力显著增强(P<0.05)。结论:pLVX-puro-myc-tagged-Twist慢病毒质粒构建成功,转录因子Twist通过MCF-7细胞上皮间质转化促进了乳腺肿瘤侵袭能力增强。

Objective ：To construct lentivirus expression vector containing Twist gene and to explore its overexpression induced epithelial to mesenchymal transition（EMT） in MCF-7 of breast cancer cells. Methods ：pcDNA3/myc-tagged-Twist plasmid was amplified by PCR and then cloned into pLVX-puro-vector. After the construction being transfected into HEK-293 cells, the supernatant was collected to infect the MCF-7 cells. The expression of Twist E-cadherin and Vimentin was analyzed by immunofluorescence and Western blot. The invasive ability was evaluated by Transwell invasion assay in MCF-7-Twist cells. Results：The pLVX-puro-myctagged-Twist was confirmed correctly by DNA sequencing. Compared with that of vector control cells, the expression of Twist, E-cadherin and Vimentin was much higher and the invasive ability was much stronger in MCF-7-Twist cells（P〈O.05）. Conclusions ：The recombinant lentivirus vector expressing Twist is constructed and is efficaciously expressed in MCF-7 cells,which could significantly promote the invasion ability of MCF-7 cells through EMT.