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目的:研究siRNA对人骨肉瘤U20S细胞MDM2基因表达及肿瘤细胞增殖的抑制作用。方法:构建可表达针对MDM2的siRNA质粒(PGCsilencerTM-MDM2-siRNA)。转染siRNA MDM2(简称siMDM2),阴性对照质粒到U20S,并设只加转染试剂作空白对照组,用逆转录聚合酶链反应(RT-PCR)和Western blotting法检测siMDM2对MDM2基因和蛋白表达的抑制作用,并用MTT法检测siMDM2对细胞增殖的抑制作用。结果:RT-PCR结果显示,转染siMDM2-1和-2组MDM2的mRNA表达量分别下调到空白对照组的32.61%和39.06%;Western blotting结果显示,转染siMDM2-1和-2组蛋白表达量下调到空白对照组的35.76%和42.20%;转染阴性对照质粒的MDM2基因和蛋白与转染试剂组比较差异均无显著性(P>0.05)。MTT结果显示,转染siMDM2后细胞生长受到明显抑制,与转染试剂组比较差异具有显著性(P<0.05),阴性对照组抑制率与转染试剂组比较差异无显著性(P>0.05)。结论:siRNA可以有效地抑制U20S细胞中MDM2的表达,并抑制细胞增殖。
Objective: To study the inhibitory effect of siRNA on MDM2 gene expression and tumor cell proliferation in human osteosarcoma U20S cells. Methods: The siRNA plasmid (PGCsilencerTM-MDM2-siRNA) targeting MDM2 was constructed. Transfection of siRNA MDM2 (siMDM2), negative control plasmid to U20S, and set only transfected reagent as a blank control group, using reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting detection of siMDM2 MDM2 gene and protein The inhibitory effect of siMDM2 on cell proliferation was detected by MTT assay. Results: RT-PCR results showed that the mRNA expression levels of MDM2 transfected with siMDM2-1 and -2 were down-regulated to 32.61% and 39.06% of the blank control group respectively. Western blotting showed that the mRNA expression of siMDM2-1 and -2 The expression levels of MDM2 gene and protein in transfected negative control plasmids were not significantly different from that in transfection reagent group (P> 0.05). The results of MTT showed that the cell growth was significantly inhibited after transfected with siMDM2, and the difference was significant compared with the transfection reagent group (P <0.05). There was no significant difference between the negative control group and the transfection reagent group (P> 0.05) . Conclusion: siRNA can effectively inhibit MDM2 expression in U20S cells and inhibit cell proliferation.