慢病毒介导MAT2A及2B双重小干扰RNA对肝癌的抑制作用

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(dual siRNA)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MAT2B mRNA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.

Growth-inhibition in hepatocelluar carcinoma caused by lentivirus mediated dual siRNA targeting MAT2A and MAT2B

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-transfected into 293T cells with the help of lipofectamine2000. 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 cytometry. The changes of S-adenosylmethionine in HepG2 cells 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 lentivirus can simultaneously inhibit the expression of MAT2A and MAT2B, resulting in growth-inhibition of hepatic cancer cells. MAT2A and MAT2B may be new thera-py targets for hepatocelluar carcinoma in the future.