Objective: To study the transcriptional regulation of SP1 on the scaffold protein ARRB1 and its influence on the progression of T-cell acute lymphoblastic leukemia (T-ALL).
Methods: pGL3-ARRB1-luc, pCDNA3.1-SP1 and other transcription factor plasmids that might be combined were constructed, and the binding of transcription factors to the promoter of ARRB1 was identified by dual luciferase reporter gene assay. Stable cell lines with over-expressed SP1 (JK-SP1) was constructed by lentiviral transfection, and the expression correlation of SP1 with ARRB1 was demonstrated by RT-PCR and Western blot. Further, the apoptosis, cell cycle and reactive oxygen species (ROS) were detected by flow cytometry. The effect of SP1 on propagation of leukemic cells was observed on NCG leukemic mice.
Results: The expression of fluorescein were enhanced by co-transfection with pCDNA3.1-SP1 and pGL3-ARRB1-luc plasmids in HEK293T cell line (P<0.001), meanwhile, compared with the control group, the expression of ARRB1 mRNA and protein were increased in JK-SP1 cells (both P<0.01). Further in vitro experiments showed that, compared with the control group, the apoptosis rate was higher (x=22.78%) , the cell cycle was mostly blocked in G1 phase (63.00%), and the content of reactive oxygen species increased in JK-SP1 cells. And in vivo experiments showed that the mice injected with JK-SP1 cells through tail vein had a favorable overall survival time (average 33.8 days), less infiltration in liver and spleen tissue.
Conclusion: Transcription factor SP1 promotes the transcription and expression of ARRB1 by binding the the promoter of ARRB1 directly, thus delays the progress of T-ALL in vitro and in vivo. The study improves the pathogenesis of ARRB1 regulating the initiation and development of T-ALL, and provides theoretical basis for the development of new possible targeted drugs.
题目: 转录因子SP1对急性T淋巴细胞白血病进程的影响.
目的: 研究转录因子SP1对支架蛋白ARRB1的转录调节及其在急性T淋巴细胞白血病（T-ALL）中的作用.
方法: 构建pGL3-ARRB1-luc、pCDNA3.1-SP1及其他可能结合的转录因子质粒，采用双荧光素酶报告基因实验证明ARRB1启动子区与转录因子结合；利用慢病毒感染构建SP1过表达的稳定细胞株JK-SP1，RT-PCR及Western blot验证SP1与ARRB1表达的关系；进一步流式细胞术检测SP1对细胞凋亡、细胞周期以及细胞活性氧含量的作用。构建NCG小鼠异种移植模型，探讨SP1对白血病小鼠成模能力的影响.
结果: pGL3-ARRB1-luc、pCDNA3.1-SP1质粒共转入HEK293T细胞后，虫荧光素高表达（P<0.001）；同时，与对照组相比，稳定细胞株JK-SP1中ARRB1 mRNA水平、蛋白水平均增加（均P<0.01）。进一步体外实验结果显示，与对照组相比，JK-SP1细胞凋亡比例更高（x=22.78%），细胞周期多阻滞于G1期（63.00%），活性氧含量增加。体内实验证明，尾静脉输注JK-SP1细胞的NCG小鼠生存时间更长（平均33.8 d），肝脾肿瘤细胞浸润相对较少.
结论: 转录因子SP1通过直接结合于ARRB1启动子区，促进ARRB1转录与表达，进而延缓体内、体外T-ALL疾病进程。本研究完善了ARRB1调控T-ALL进程的机制并为新的靶向药物研发提供了理论依据.
Keywords: ARRB1; SP1; T-cell acute lymphoblastic leukemia.