骨桥蛋白siRNA在肝癌HepG2细胞中的沉默效应

Osteopontin silencing by small interfering RNA in HepG2 cells

目的观察骨桥蛋白(OPN)RNA干扰(siRNA)在肝癌HepG2细胞中的沉默效应。方法设计合成骨桥蛋白RNA干扰寡核苷酸片段,将之克隆至pGCsi真核表达载体上,并经测序分析鉴定。用脂质体法将质粒转染HepG2细胞筛选稳定转染OPNsiRNA的细胞,并经Westernblot鉴定。结果成功构建3个pGCsiOPN质粒,瞬时转染293T细胞后发现pGCsiOPN2有干扰效应;进一步稳定转染肝癌HepG2细胞,筛选到4株阳性细胞克隆。结论OPNsiRNA在肝癌HepG2细胞中有沉默效应。

Objective To investigate if osteopontin（OPN） expression was suppressed by small interfering RNA（siRNA） in HepG2 cells. Methods Three plasmids containing OPN-siRNA fragments were constructed by gene recombination with vector pGCsi and OPN-siRNA oligonucleotides. Lipofectamine was used to transfect 293T and HepG2 cells. Western blot was used to quantify OPN protein levels. Results We successfully constructed three plasmids of pGCsi-OPN1, pGCsi-OPN2 and pGCsi-OPN3 ,and found that OPN gene was inhibited when pGCsi-OPN2 was used to transfect 293T cells. After transfection of HepG2 cells with pGCsi-OPN2, we generated 4 cell lines stably expressing pGCsi-OPN2 in HepG2 cells, and found that OPN expression was down-regulated in these cells. Conclusion This study demonstrates that RNA interference stably reduces the expression of OPN in HepO2d cells.