人Bub1基因shRNA真核表达质粒的构建及其对人卵巢癌细胞SKOV3紫杉醇敏感性的影响

Construction of pEGFP-Bub1-shRNA Recombinant Plasmid and Its Effect on the Sensitivity of SKOV3 Cells to Paclitaxel

目的构建人Bub1 shRNA真核表达质粒,鉴定该质粒对人卵巢癌SKOV3细胞中Bub1基因的抑制作用,并探讨其对SKOV3细胞紫杉醇敏感性的影响。方法构建pEGFP-Bub1-shRNA质粒,将其经脂质体Lipo 2000TM转染SKOV3细胞后,通过RT-PCR及Western blot检测干扰效率,流式细胞仪检测细胞凋亡率,MTT法比较转染前后SK-OV3细胞的紫杉醇敏感性。结果成功构建了pEGFP-Bub1-shRNA质粒;RT-PCR和Western blot检测结果提示,转染后Bub1的mRNA和蛋白表达均显著降低;MTT和FACS提示,与对照组细胞相比,转染后SKOV3细胞生长抑制率及凋亡率明显降低(P<0.05)。结论pEGFP-Bub1-shRNA质粒能有效抑制Bub1 mRNA及蛋白的表达,并降低SK-OV3细胞紫杉醇敏感性,该质粒的成功构建为进一步研究Bub1基因的生物学功能提供了工具。

Objective To construct the short hairpin RNA（shRNA） expression vector specific to human Bub1 gene and investigate its silence effect on Bub1 gene,and then explore the function of Bub1.Methods pEGFP-Bub1-shRNA was designed and constructed,and then transfected into human ovarian carcinoma cell line SKOV3.The expression change of Bub1 was examined by RT-PCR and Western blot.Inhibition of cell proliferation was determined by MTT assay,and the induced apoptosis was analyzed by FACS.Results RT-PCR and Western blot revealed that pEGFP-Bub1-shRNA/SKOV3 cells expressed lower level of Bub1 mRNA and protein after transfection with pEGFP-Bub1-shRNA plasmid than the controls.The sensitivity of pEGFP-Bub1-shRNA/SKOV3 cells to paclitaxel was lower than SKOV3 cells and pEGFP-C1/SKOV3 cells.Conclusion Transfection of the pEGFP-Bub1-shRNA plasmid could decrease the expression level of Bub1,which could reduce paclitaxel sensitivity in human ovarian cancer cell line SKOV3.