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

摘要:

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-puro-myc-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。

Abstract:

Objective：To construct lentivirus expression vector containing Twist gene and to explore its overexpression induced epithe－lial 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-myc-tagged-Twist was confirmed correctly by DNA sequencing. Compared with that of vector control cells，the expression of Twist，E-cad－herin and Vimentin was much higher and the invasive ability was much stronger in MCF-7-Twist cells（P<0.05）. 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.