干扰素应答因子1对不同p53状态骨肉瘤凋亡的调节作用

The effect of interferon regulatory factor-1 on apoptosis regulation in osteosarcomawith differentp53 status

目的观察干扰素应答因子1（IRF-1）基因对不同p53状态骨肉瘤细胞凋亡的调节作用，并探讨其机制。方法利用u20s（p53^＋/＋）和Saos2（p53^-/-）骨肉瘤细胞作为研究模型，用不同剂量阿霉素处理后，Western blot检测IRF-1表达的变化。进一步通过过表达IRF-1基因或敲除IRF-1基因，检测其对骨肉瘤细胞凋亡的影响。并通过实时定量逆转录聚合酶链反应（RT-qPCR）法检测IRF-1对p53下游靶基因B细胞淋巴瘤/白血病-2相关x蛋白（bax）和p21的调节作用。结果不同剂量阿霉素处理U20S和Saos2后，Westernblot结果显示IRF-1的表达明显增高。进一步过表达IRF-1后，乳酸脱氢酶（LDH）法检测细胞死亡率明显增高（P〈0．05）；干涉IRF-1基因后，全量LDH检测细胞死亡率则明显降低（P〈0．05）。RT-qPCR检测显示，IRF-1对p53下游靶基因bax和p21在p53^-/-SaOS-2细胞中具有明显的促进作用（P〈0．05），而对于p53^＋/＋U20S中的作用并不明显（P〉0．05）。结论IRF-1可通过调节p53下游靶基因bax和p21的表达，从而显著促进阿霉素诱导的骨肉瘤细胞凋亡。

Objective Detect the effects of interferon regulatory factor-1 （ IRF-1 ） gene on the ap optosisregulation in osteosarcoma with different p53 status and disclose the underlying mechnisms prelimina rily. Methods To observe the effects of IRF-1 gene on the apoptosis effects induced by chemotherapeutic agent adriamycin in steosarcoma with different p53 status, p53^＋/＋ U2OS and p53^-/-SaOS-2 were used. Western blotting analysis was firstly used to detect the expression of IRF-1 protein in adriamycin treated p53 ^＋/＋ U2OS and p53^ -/- SaOS-2 cells. And then IRF-1 gene was further overexpressed or knocked out for further detection the apoptosis effect induced by adriamycin respectively. Meanwhile, the target genes of 1953, B cell lymphoma/leukemia-2 associated X protein （bax） and p21, were detected by real-time reverse transcriptase-polymerase chain reaction （RT-qPCR）. Results Western blotting showed that the expres sion of IRF-1 has been improved significantly when p53 ^＋/＋ U2OS and p53 ^-/- SaOS-2 were treated with ad riamycin for 24 h. The significant increase in the death rate of cells has been detected by the release of lac tate dehydrogenase （ LDH ） Activity （ P 〈 0. 05 ）. When IRF-1 gene were overexpressed with transient transfection, adriamycin-induced apoptosis effect were increased significantly （P 〈 0. 05 ）, while the knockdown of IRF-1 gene by small interfering RNA （ siRNA ） transfection were decreased significantly （ P 〈 0.05 ）. RT-qPCR were used to further detected the expression of downstream target genes bax and p21, which do great favor to p53-dependent apoptosis. The results showed that bax and p21 were both up- regulated significantly in p53^-/-osteosarcoma cells （P 〈 0. 05 ） , but not observed in p53 ^＋/＋ osteosarcoma cells （ P 〉 0.05 ）. Conclusion IRF-1 plays a crucial role in adrymycin induced apoptosis by modulating p53 target genes, especially to the cancer cells with p53 gene deficient.