摘要
目的观察慢病毒载体携带的针对MAT2A和MAT2B基因双重小干扰RNA（dualsiRNA）对肝癌的抑制作用。方法分别构建靶向MAT2A、MAT2B基因的siRNA质粒，酶切、连接，构建同时针对MAT2A和MAT2B基因的慢病毒载体siMAT2A／2B，感染肝癌HepG2细胞，逆转录-聚合酶链反应（RT-PCR）测定MAT2A和MAT2BmRNA水平，细胞计数；流式细胞仪测定细胞凋亡，测定作用前后肝癌细胞内S-腺苷甲硫氨酸（SAMe）的水平。结果成功构建慢病毒载体携带的针对MAT2A和MAT2B基因双重小干扰RNA，LV—siMAT2A／2B同时抑制肝癌MAT2A和MAT2B基因表达分别达到89．5％和97．8％，肝癌HepG2细胞生长抑制率大于50％，促进细胞凋亡，提高肝癌细胞内SAMe的水平上升了2倍。结论同时抑制肝癌内MAT2A、MAT2B基因的表达可以抑制肝癌的生长。
Objective To investigate the suppressive effects caused by lentivirus mediated dual siRNA targeting MAT2A and MAT2B expression in hepatic cancer HepG2 cells. Methods Two siRNAs targeting MAT2A and MAT2B respectively were cloned to one lentivirus work vector simultaneously. Work vector and three package plasmids were co-transfeeted into 293T cells with the help of lipofeetamine2000. Lentivirus was collected after 72 h, and was added to the cultured HepG2 cells. Cells were counted every day and total mRNA was extracted and underwent RT-PCR. Apoptosis was figured out with flow eytometry. The changes of S-adenosylmethionine in HepG2 ceils were also studied. Results Dual small interfering RNA targeting MAT2A and MAT2Bs imultaneously was constructed successfully. Expression of MAT2A and MAT2B was suppressed by 89.5% and 97.8% respectively. Cell growth was inhibited by 50%. Intra- cellular S-adenosylmethionine level was increased over 2 times. Cell apoptosis was induced. Conclusion Dual small interfering RNA mediated by lenfivirus can simultaneously inhibit the expression of MAT2A and MAT2B, resulting in growth-inhibition of hepatic cancer cells. MAT2A and MAT2B may be new therapy targets for hepatocelluar carcinoma in the future.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第2期184-186,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（30872491）
关键词
癌
肝细胞
慢病毒
RNA干扰
Carcinoma,hepatocelluar
Lentivirus
RNA interfering
