靶向上皮性卵巢癌SKOV3细胞ZEB1-shRNA重组体构建及功能研究

Construction of targeting ZEB1-shRNA recombinant and its functional research in epithelial ovarian cancer SKOV3 cells

目的:利用RNA干扰技术,降低人上皮性卵巢癌SKOV3细胞中锌指增强子结合蛋白1(ZEB1)基因的表达,观察ZEB1低表达对SKOV3细胞的生长影响。方法:用pSUPER-EGFP1载体构建针对人ZEB1基因的干扰质粒shZEB1,脂质体转染SKOV3细胞,G418筛选稳定转染细胞株,通过RT-PCR和Western blot检测ZEB1在SKOV3细胞中表达。用克隆形成、划痕试验、RT-PCR和致瘤试验分别检测转染细胞克隆能力、迁移力、miR-200c表达水平和在裸鼠的致瘤性。结果:成功构建shZEB1,稳定转染细胞株shZEB1-SKOV3内ZEB1表达下降,导致shZEB1-SKOV3细胞miR-200c表达增高,克隆能力、迁移力及致瘤性下降。结论:用RNA干扰技术可靶向降低卵巢癌细胞株SKOV3内ZEB1的表达,使其致瘤性降低,提示ZEB1可作为分子靶点用于上皮性卵巢癌靶向治疗。

Objective：To construct the recombined plasmid expressing zinc-finger E-box binding homeobox 1 （ZEB1） short hairpin RNA （shRNA） and observe the characteristics of epithelial ovarian cancer SKOV3 cells after shRNA-mediated ZEB1 gene silencing in an epithelial ovarian cancer SKOV3 cells. Methods ：The shRNA targeting the ZEB1 gene was synthesized, and cloned into plasmid pSUPER-enhanced green fluorescent protein 1 （EGFP1）. The formed pSUPER-EGFP1-ZEBI-shRNA was transfeeted into the ovarian cancer SKOV3 cells by Lipofeetamine 2000, and the stably transfected cells were isolated by G418 selection. The expression of ZEB1 was detected by RT-PCR and Western blot. The characteristics of ZEBI-shRNA transfected SKOV3 cells were analyzed from the assays of colony formation, wound healing and over expression of miR-200c as well as tumorigenicity in nude mice, respectively. Results：The ZEB1 expression in ZEBI-shRNA transfected SKOV3 cells was significantly lower than that of the scramble control siRNA. The ZEBI-shRNA transfeeted SKOV3 cells show the abilities of proliferation, migration and tumorigenicity were significantly decreased compared with the scrambled control siRNA（ P 〈 0.05 ）. ZEB1 low expression resulted in increased miR-200c expression in the ZEB1- shRNA transfected SKOV3 cells. Conclusion：Targeting ZEB1 expression in SKOV3 cells with RNA interference can significantly reduce and inhibit the cell proliferation and the tumorigenicity of SKOV3 cells. These results suggest that the ZEB1 is an effective therapeutic target for human epithelial ovarian caner treatment.