Xiong Jian-jun, Gan Li-jun, Lin Ling, Gong Zhen, Wu Zhou-huan, Li Wei-dong
Department of Pharmacology, College of Basic Medical Science, Jiujiang University, Jiujiang 332000, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2011 Mar;42(2):166-9.
To construct eukaryotic expression plasmid expressing siRNA targeting ubiquitin specific peptidase 22 gene (USP22), and to investigate its effect on the growth of hepatoma carcinoma cells HepG2.
siRNA templates were synthesized based on USP22 mRNA sequence and cloned into vector Pmscv/Hyg/U6. The resulting recombinant was identified by restriction enzyme digestion and DNA sequencing. Recombinants were than transfected into HepG2 cells mediated by liposome. The USP22 protein and mRNA in HepG2 cells were detected by western blot and RT-PCR, respectively. The cellular growth activity was evaluated with MTT assay.
Recombinant plasmid expressing siRNA targeting USP22 was successfully constructed. The down-regulated protein and mRNA level of USP22 and decreased cellular growth in HepG2 cells transfected with recombinant plasmid were observed.
The eukaryotic expression vector for RNA interference USP22 gene is constructed successfully, which inhibits the expression of USP22 in HepG2 cells and suppresses cell proliferation.
构建靶向泛素特异性肽酶22基因（USP22）的小干扰RNA（siRNA）真核表达质粒，并研究其对肝癌细胞HepG2生长的影响。
根据USP22 mRNA序列合成siRNA模板，克隆至载体Pmscv/Hyg/U6。通过限制性内切酶酶切及DNA测序鉴定重组体。然后将重组体经脂质体介导转染至HepG2细胞。分别采用蛋白质免疫印迹法和逆转录-聚合酶链反应检测HepG2细胞中USP22蛋白和mRNA水平。采用MTT法评估细胞生长活性。
成功构建了靶向USP22的siRNA重组表达质粒。观察到转染重组质粒的HepG2细胞中USP22蛋白和mRNA水平下调，细胞生长受抑制。
成功构建了RNA干扰USP22基因的真核表达载体，其可抑制HepG2细胞中USP22的表达并抑制细胞增殖。
