Tang Shi, Wang Hao-Biao, Guo Wei, Zou Lin, Liu Shan
Center for Clinical Molecular Medicine, Children's Hospital Affiliated to Chongqing Medical University,Chongqing 400014, China.National Clinical Research Center for Child Health and Disorders,Chongqing 400014, China.Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing 400014, China.Chongqing Key Laboratory of Pediatrics, Chongqing 400014, China.
Clinical Research Unit, Children's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200062, China.E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2023 Feb;31(1):57-63. doi: 10.19746/j.cnki.issn.1009-2137.2023.01.009.
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).
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 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.
The expression of fluorescein were enhanced by co-transfection with pCDNA3.1-SP1 and pGL3-ARRB1-luc plasmids in HEK293T cell line (0.001), meanwhile, compared with the control group, the expression of ARRB1 mRNA and protein were increased in JK-SP1 cells (both 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 G 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.
Transcription factor SP1 promotes the transcription and expression of ARRB1 by binding the the promoter of 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.
研究SP1对支架蛋白ARR B1的转录调控及其对T细胞急性淋巴细胞白血病（T-ALL）进展的影响。
构建pGL3-ARRB1-luc、pCDNA3.1-SP1及其他可能相互结合的转录因子质粒，通过双荧光素酶报告基因检测法鉴定转录因子与ARRB1启动子的结合情况。采用慢病毒转染构建SP1过表达稳定细胞系（JK-SP1），通过RT-PCR和蛋白质免疫印迹法验证SP1与ARRB1的表达相关性。进一步采用流式细胞术检测细胞凋亡、细胞周期及活性氧（ROS）水平。在NCG白血病小鼠体内观察SP1对白血病细胞增殖的影响。
在HEK293T细胞系中，共转染pCDNA3.1-SP1和pGL3-ARRB1-luc质粒可增强荧光素表达（P<0.001）；同时，与对照组相比，JK-SP1细胞中ARRB1 mRNA和蛋白表达均升高（均P<0.01）。进一步体外实验表明，与对照组相比，JK-SP1细胞凋亡率更高（χ²=22.78%），细胞周期大多阻滞于G期（63.00%），活性氧含量增加。体内实验显示，经尾静脉注射JK-SP1细胞的小鼠总生存时间良好（平均33.8天），肝脾组织浸润较少。
转录因子SP1通过直接结合ARRB1启动子促进其转录表达，进而在体内外延缓T-ALL进展。本研究完善了ARRB1调控T-ALL发生发展的发病机制，为开发新的潜在靶向药物提供了理论依据。
