siRNA抑制肝癌细胞甲硫氨酸腺苷转移酶2A基因表达

Inhibition of MAT 2A gene expression in hepatoma cell by short interfering RNAs

目的构建siRNA体内表达载体pSilence-2.1-U6-siRNA,筛选出抑制甲硫氨酸腺苷转移酶(MAT)2A表达的有效靶序列,为MAT2A的功能研究奠定了基础。方法以MAT2A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plucF-MAT2A。脂质体转染法将重组质粒载体plucF-MAT2A与产生siRNA的质粒pSilence-2.1-U6共转染293T细胞,定量测量荧光素酶活性,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(RT-PCR)检测MAT2AmRNA表达,并检测转染后肝癌细胞MAT的活性及细胞凋亡情况,进一步证实siRNA对MAT2A表达的抑制效果。结果所合成的4条小干扰RNA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MAT2AmRNA表达,降低了肝癌细胞中MAT活性,诱导肝癌细胞凋亡。结论siRNA抑制肝癌细胞MAT2A基因表达。

Objective To construct the expression vector pSlience-2.1-U6-siRNA that can express the short interfering RNAs (siRNA) against MAT2A gene and screen the effective target sequence of siRNA.Methods The four siRNA against MAT2A gene were transcript synthesized intracelluarly by expressed templates of plasmid vector pSilence-2.1-U6,and the target sequence of MAT2A gene was inserted into the upstream of the reporter gene in order to construct the recombinant plasmid vector plucF-MAT2A.The recombinant plasmid and siRNA -producing plasmid were cotransfected into 293 T cells by using Lipofectamine methods.The luciferase activity in cell lysate was quantitatively measured and the effective siRNA inhibiting the expression of luciferase was screened out.After the effective siRNA was transfected into Bel-7402 cells,the expression of MAT2A mRNA was semiquantitatively detected by semiquantitative RT-PCR.The MAT activity of hepatoma cells and apoptosis after transfection was measured.Results Two siRNA among the 4 synthesized siRNA displayed inhibitory effects on the lucifermase expression with the inhibitory rate being 81% and 89% respectively.The expression of MAT2A mRNA in Bel-7402 cells was specially ihhibited and the MAT activity decreased,and ultimately hepatic apoptosis induced.Conclusion The siRNA can inhibit the expression of MAT2A gene in hepatocellular carcinoma.