Mao Wei-wei, Xiong Peng, Han Feng, Hu Zhi-jian
Department of Laboratory Medicine, Faculty of Clinical Medicine, Jiujiang, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 Sep;28(9):985-7.
To construct an eukaryotic expression vector for RNA interference targeting activating transcription factor 2 (ATF-2) gene, and explore its effect on proliferation and apoptosis of HepG2 cells.
Two complementary oligonucleotides were synthesized based on ATF-2 mRNA sequence. The annealed fragment was inserted into the vector PBA-siU6. The recombinant plasmid PBA-siATF-2 was confirmed by DNA sequencing and transfected into HepG2 cells mediated by liposome. After transfection, ATF-2 protein was detected by Western blotting. The cellular growth activity and apoptosis rate were measured by MTT assay and flow cytometry, respectively.
Recombinant plasmid expressing siRNA targeting ATF-2 gene was confirmed by DNA sequencing. Plasmid transfection down-regulated the level of ATF-2 protein in HepG2 cells, which blocked cellular growth and induced cell apoptosis.
The eukaryotic expression vector for RNA interference targeting ATF-2 gene was constructed successfully, which inhibits HepG2 cell proliferation and induces cell apoptosis.
构建针对激活转录因子2（ATF - 2）基因的RNA干扰真核表达载体，并探讨其对肝癌细胞系HepG2细胞增殖和凋亡的影响。
根据ATF - 2 mRNA序列合成两条互补寡核苷酸。退火后的片段插入载体PBA - siU6中。通过DNA测序确认重组质粒PBA - siATF - 2，并通过脂质体介导转染至HepG2细胞。转染后，采用蛋白质免疫印迹法检测ATF - 2蛋白。分别采用MTT法和流式细胞术检测细胞生长活性和凋亡率。
通过DNA测序确认了表达针对ATF - 2基因的小干扰RNA的重组质粒。质粒转染下调了HepG2细胞中ATF - 2蛋白水平，抑制细胞生长并诱导细胞凋亡。
成功构建了针对ATF - 2基因的RNA干扰真核表达载体，其可抑制HepG2细胞增殖并诱导细胞凋亡。
