载体介导的RNA干扰技术抑制肝癌细胞中糖原合成酶激酶3β表达的研究  

Vector-mediated RNA interference of GSK-3β expression in hepatoma cell

作　　者：张娜[1] 刘璐[1] 窦玥莹 王真[1] 宋丹青[1] 李电东[1] 邓洪斌[1] 

机构地区：[1]中国医学科学院北京协和医学院医药生物技术研究所生化室,北京100050

出　　处：《中国医药生物技术》2015年第2期131-138,共8页Chinese Medicinal Biotechnology

基　　金：北京协和医学院"协和青年基金"(3332013144);国家自然科学基金面上项目(81473248;81170326)

摘　　要：目的构建和筛选能高效、特异性抑制人GSK-3β基因表达的siRNA表达载体,探讨其对肝癌细胞增殖和凋亡的影响。方法提取人L02细胞总RNA,RT-PCR扩增GSK-3β基因序列,克隆至pSEB-HUS真核表达载体,构建pSEB-G重组质粒。将设计、合成的3条靶向GSK-3β基因编码区的siRNA及阴性对照分别克隆至pSEB-G,构建siRNA表达载体pSEB-si1-G、pSEB-si2-G、pSEB-si3-G及pSEB-siN-G。将siRNA表达载体瞬时转染HepG2细胞,通过绿色荧光信号、RT-PCR和Western blot观察和检测其对GSK-3β基因的抑制效果,MTT实验和caspase-3活性分析检测其对HepG2细胞增殖和凋亡的影响。结果经双酶切及测序证实,所构建si RNA表达载体目的基因大小、序列与预期相符。瞬时转染HepG2细胞后,其中的pSEB-si1-G能使细胞中绿色荧光信号明显减少,可显著抑制GSK-3βmRNA的表达及蛋白的合成,并使HepG2细胞增殖明显降低,caspase-3活性显著升高。结论成功构建了靶向GSK-3β基因的siRNA表达载体,沉默GSK-3β能显著抑制HepG2细胞的生长并诱导其发生凋亡。Objective To construct a vector expressing siRNA targeting human GSK-3β gene and to investigate its effects on proliferation and apoptosis of HepG2 cells.Methods The c DNA of GSK-3β was amplified by RT-PCR from the total RNA of L02 cells, and then inserted into p SEB-HUS vector to generate pSEB-G plasmid. The synthesized si RNAs targeting the coding region of human GSK-3β were cloned into p SEB-G respectively, resulting in the pSEB-si1-G, pSEB-si2-G, p SEB-si3-G and p SEB-si N-G plasmids. These plasmids were transiently transfected into HepG2 cells, and the expression levels of GSK-3β were detected by green fluorescent signal, RT-PCR, and Western blot. The effects of si RNAs on proliferation and apoptosis of Hep G2 cells were determined by MTT assay and caspase-3 activity assay. Results All plasmids were successfully constructed as confirmed by restriction enzyme digestion and DNA sequencing. The pSEB-si1-G vector had the most significant knockdown effect on GSK-3β among the constructed plasmids. GSK-3β silencing resulted in marked decrease of proliferation and increase of caspase-3 activity in HepG2 cells. Conclusion A si RNA expression vector targeting GSK-3β is successfully constructed and validated for its knockdown effect, and silencing of GSK-3β can significantly inhibit HepG2 cell proliferation and induce its apoptosis.

关 键 词：糖原合成酶激酶3Β RNA干扰 癌 肝细胞 SIRNA表达载体 

分 类 号：R735.7[医药卫生—肿瘤]