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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 siRNA expression vector pSilence-2.1-U6-siRNA and screen the effective target sequence which inhibits the expression of methionine adenosine transaminase (MAT) 2A, which lays the foundation for the functional study of MAT2A. Methods MAT2A was used as the target gene to generate the siRNA plasmid vector pSilence-2.1-U6 as the expression template, and four siRNAs were synthesized by intracellular transcription. The recombinant plasmid vector plucF-MAT2A carrying the luciferase reporter gene was constructed. Lipofectamine was transfected into 293T cells by co-transfection of recombined plasmid vector plucF-MAT2A and siRNA-producing plasmid pSilence-2.1-U6, luciferase activity was measured quantitatively, and effective siRNAs for inhibiting luciferase expression were initially screened, then Effective siRNA transfected Bel-7402 hepatoma cells, MAT2A mRNA expression was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), MAT activity and apoptosis were detected, further confirmed that siRNA on MAT2A Inhibitory effect of expression. Results Two of the four small interfering RNAs were synthesized and their inhibitory activities were 81% and 89%, respectively. Two siRNAs specifically inhibited the expression of MAT2A mRNA in hepatocellular carcinoma cells, decreased the activity of MAT in hepatoma cells and induced the apoptosis of hepatoma cells Death. Conclusions siRNA inhibits MAT2A gene expression in hepatocellular carcinoma cells.