摘要
目的研究靶向甲硫氨酸腺苷转移酶(MAT)2A基因的小干扰RNA(siRNA)对肝癌细胞生长和细胞凋亡的影响。方法以MAT 2A为目的基因,以产生siRNA质粒载体pSilence-2.1-U6为表达模板,细胞内转录合成4条siRNA;并构建携带荧光素酶报告基因的重组质粒载体plucA-MAT 2A。脂质体转染法将重组质粒载体plucA-MAT 2A与产生siRNA的质粒pSilence-2.1-U6共转染293 T细胞,定量检测荧光素酶活性,初步筛选出抑制荧光素酶表达的有效siRNA,然后将有效的siRNA转染Bel-7402肝癌细胞,半定量逆转录聚合酶链反应检测MAT 2A mRNA表达,并检测转染后肝癌细胞MAT的活性,进一步采用四甲基偶氮唑盐法观察siRNA对肝癌细胞生长的抑制率,用流式细胞仪检测siRNA对肝癌细胞凋亡的影响。结果所合成的4条siRNA中有2条抑制荧光素酶表达,抑制效率分别为81%和89%,并特异性抑制肝癌细胞MAT 2A表达,降低了肝癌细胞中MAT活性,抑制肝癌细胞的生长,诱导肝癌细胞凋亡。结论靶向MAT 2A基因的siRNA抑制肝癌细胞生长,诱导肝癌细胞凋亡;MAT 2A是肝癌基因治疗的一个很有希望的靶位点。
Objective To investigate the effect of short interfering RNA targeting MAT 2A on growth and apoptosis of hepatoma cells. Methods The four siRNA against MAT 2A gene were transcript synthesized intracelluarly by expressed templates of plasmid vector pSilence-2.1-U6. We inserted the target sequence of MAT 2A gene into the upstream of the reporter gene in order to construct the recombinant plasmid vector plucA- MAT 2A. The recombinant plasmid and siRNA-producing plasmid were co-transfected into 293 T cells using this construct via lipofectamine methods. The inhibition effect was detected by measuring luciferase activity in the cell lysate to screen the effective siRNA, and then, the effective siRNA was transfected into Bel-7402 cells. The effect of siRNA treatment on the MAT 2A mRNA level and the MAT activity of hepatoma cells were measured. In order to study the effect of short interfering RNA targeting MAT 2A on growth and apoptosis of hepatoma cells, the tumor cell killing rate was analyzed by MTT method and the rate of apoptosis of hepatoma cells was evaluated by flow cytometry. Results The two siRNA among the four siRNA displayed inhibitory effect on the lucifermase expression with the inhibitory rates of 81% and 89% respectively. The expression of MAT 2A mRNA in Bel-7402 cells was specifically inhibited and the MAT activity in Bel-7402 cells was decreased. Furthermore, silencing of the MAT 2A gene by RNAi significantly inhibited hepatoma cell growth and led to induction of apoptosis. Conclusion RNA interference-mediated silencing of MAT 2A gene attenuates growth and induces apoptosis of hepatoma cells; MAT 2A is an ideal target of gene-specific therapy for liver cancer.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2005年第5期335-338,共4页
Chinese Journal of Hepatology
