联合转染Livin和Survivin shRNA表达载体对肝癌HepG2细胞生物学行为的影响

目的:分别构建Livin、Survivin基因短发夹RNA(shRNA)真核表达载体,探究联合转染Livin和Survivin真核表达载体对肝癌HepG2细胞生物学行为的影响。方法:分别设计并构建针对Livin、Sur-vivin基因shRNA真核表达载体pSD11-Livin和pSD11-Survivin,脂质体法单独或联合转染肝癌HepG2细胞,分空白对照组、质粒对照组、Survivin组、Livin组和共转染组。荧光定量PCR、Western blot分别检测Livin、Survivin mRNA及蛋白的相对表达量,MTT法检测细胞增殖的变化,TUNEL法检测细胞凋亡率。结果:针对Livin、Survivin基因的shRNA真核表达载体构建成功。共转染组Livin、SurvivinmRNA相对表达量分别为0.120±0.022、0.325±0.125,与单独转染组相比显著降低(P<0.05);共转染组Livin、Survivin蛋白相对表达量分别为0.412±0.099、0.473±0.051,与单独转染组相比差异具有统计学意义(P<0.05)。共转染组转染后48、60、72 h细胞生长抑制率均显著高于单独转染组(P<0.05),转染后48 h细胞凋亡率明显上升(P<0.05)。结论:针对Livin、Survivin基因的shRNA真核表达载体构建成功。联合转染pSD11-Livin和pSD11-Survivin更有效地降低了Livin和Survivin基因在HepG2细胞中的表达,更显著地抑制了肝癌细胞的增殖,促进了肝癌细胞的凋亡。

Impact of co-transfection of Livin and Survivin shRNA expression vector on biological behavior of HepG2 cells

Objective： To construct a short hairpin RNA （ shRNA ） eukaryotic expression vec-tor targeting Livin or Survivin gene, and explore the impact of Co-transfection of Livin and Survivin shRNA expression vector on biological behavior of HepG2 cells. Methods： shRNA eukaryotic ex-pression vector pSDt 1 -Livin and pSD11-Survivin were designed, constructed, and transfected intc HepG2 cells in combination with liposome. Cells were divided into blank control group, negative ：ontrol group, Survivin group, Livin group and co-transfection group, mRNA relative expression was detected by fluorescence quantitative PCR. Western blot were used to detect Livin, Survivin proteir respectively. Changes of cell proliferation were detected by MTT, and TUNEL was used to detecl apoptosis rate. Results＇. the Livin and Survivin shRNA eukaryotic expression vectors were suc-cessfully constructed. Livin, Survivin mRNA relative expression quantity in HepG2 cells of co-trans-fection group was 0.120 ±0.022 and 0.325 ±0.125 respectively. Compared with Survivin group or Livin group, mRNA relative expression quantity of co-transfection group was decreased significantly CP 〈 0.05）. Livin, Survivin protein relative expression quantity in HepG2 cells of co-transfection group was 0.412 ± 0.099 and 0.473 ± 0.051. Co-transfection inhibited protein expression more effi- ciently than single-transfection （P〈 0.05）. Cell growth inhibition rate in co-transfection group were higher than those in single-transfection group on 48 h, 60 h, 72 h after transfection（P〈0.05）. Apop-tosis rate increased more significantly in co-transfection group than any other groups （P 〈 0.05）. Conclusion： Livin, Survivin shRNA eukaryotic expression vector was successfully constructed. Co-transfection of pSD11-Livin and pSD11-Survivin reduced the expression of Livin and Survivin gene in HepG2 cells more effectively, inhibited the proliferation of hepatoma cells more significantly, and induced the apoptosis of HepG2 cells more effectively.