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目的 探讨干扰素α(IFN-α)调控肝癌细胞SMMC-7721胸苷磷酸化酶(TP)表达的机制.方法 应用逆转录聚合酶链反应(RT-PCR)检测SMMC-7721细胞中TP mRNA的表达水平;应用JAK-STAT信号通路阻断剂AG-490研究该通路的作用;应用基因转录抑制剂放线菌素D研究TP mRNA的半衰期.结果 IFN-α上调TP mRNA表达具有时间-剂量依赖性.10000U/rrd的IFN-α处理SMMC-7721细胞8h后,TP mRNA表达水平开始升高,至12h时峰值达0.7272±0.0582,此后维持在0.6573±0.1873水平至72h.5000U/ml和10000U/ml的IFN-α处理肝癌细胞24h,TP mRNA表达水平分别为0.5991±0.2179和0.6298±0.1786,与未用IFN-α处理的肝癌细胞(0.2231±0.0595)相比,差异有统计学意义(P<0.05).应用AG-490阻断JAK-STAT通路,IFN-α上调TP mRNA表达的能力明显受到抑制(0.2545±0.1053),与未处理的肝癌细胞(0.2329±0.1032)相比,差异不明显,仅应用IFN-α而未应用AG-490处理的肝癌细胞,其TP mRNA表达水平则显著升高(0.7209±0.1168).应用IFN-α处理SMMC-7721细胞24h后,利用放线菌素D阻断RNA聚合酶,TP mRNA半衰期为35.8h,而未用IFN-α处理的肝癌细胞为8.5h.结论 一定剂量的IFN-α上调TP mRNA的表达水平,与细胞内JAK-STAT信号通路有关;IFN-α具有转录后稳定TP mRNA的作用.

Objective To examine how the thymidine phosphorylase (TP) gene expression is upregulated by interferon-α (IFN-α) in human hepatocellular carcinoma SMMC-7721 cells. Methods TP mRNA levels were determined by RT-PCR. Whether the JAK-STAT cascade mediates IFN-a-induced TP mRNA expression was studied by pretreatment with Janus Kinase (JAK) inhibitor, AG-490. Effects of IFN-αon TP mRNA stability were detected with additional actinomycin D. Results The expression of TP mRNA was induced by IFN-α in a dose- and time-dependent manner in SMMC-7721 (human hepatocellular carcinoma) cells. TP mRNA levels rose at 8h, reached the peak value at 12h, and remained at a high level up to 72h in SMMC-7721 cells treated with IFN-α 10000U/ml. IFN-α at a dose of 5000 or 10000U/ml up-regulated TP expression about 3 fold compared with that of non-treated cells (P<0.05). Induction of TP mRNA expression by IFN-α was significantly inhibited in SMMC-7721 cells by pretreatment with AG-490, in comparison with that treated with IFN-α alone. Pretreatment of SMMC-7721 cells with IFN-α 10000U/ml for 24h caused a substantial stabilization of TP mRNA, with a half-live of 35.8h, compared with 8.5hr in the control SMMC-7721 cells. Conclusion IFN-α at certain doses upregulates TP mRNA expression via both JAK-STAT transcriptional activation and post-transcriptional mRNA stabilization in human hepatocellular carcinoma SMMC-7721 cells.