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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 effects of small interfering RNA (siRNA) targeting the methionine adenylyltransferase (MAT) 2A gene on hepatocellular carcinoma cell growth and apoptosis. METHODS: MAT 2A was used as the target gene to generate siRNA plasmid vector pSilence-2.1-U6 as the expression template, and four siRNAs were synthesized by intracellular transcription. Recombinant plasmid vector plucA-MAT 2A carrying luciferase reporter gene was constructed. Liposome transfection method The recombinant plasmid vector plucA-MAT 2A and siRNA-producing plasmid pSilence-2.1-U6 cotransfected 293T cells, quantitative detection of luciferase activity, preliminary screening of effective siRNA inhibition of luciferase expression, Then, siRNAs were transfected into Bel-7402 hepatoma cells. The mRNA expression of MAT 2A was detected by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The activity of MAT was detected by MTT assay. The inhibitory rate of hepatoma cell growth was detected by flow cytometry siRNA on hepatocellular carcinoma cell apoptosis. Results Two of the 4 siRNAs inhibited the luciferase expression and the inhibitory rates were 81% and 89%, respectively. The two siRNAs specifically inhibited the expression of MAT 2A in hepatoma cells, decreased the activity of MAT in hepatoma cells and inhibited the growth of hepatoma cells , Induce apoptosis of hepatoma cells. Conclusion siRNA targeting MAT 2A gene inhibits the growth of hepatocellular carcinoma cells and induces the apoptosis of hepatocellular carcinoma cells. MAT 2A is a promising target for gene therapy of hepatocellular carcinoma.