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目的研究在曲古菌素A(trichostatin A,TSA)处理条件下细胞周期素D2的调控机制。方法细胞周期素D2的mRNA水平通过RT-PCR方法检测。细胞周期素D2的启动子片段以A549细胞基因组DNA为模板,通过高保真PCR扩增获得,并克隆到pGL3-BASIC载体中;并以此为基础通过高保真PCR扩增出不同长度的启动子片段。启动子相对活性通过萤火虫酶报告基因方法确定。结果在TSA处理条件下,细胞周期素D2的mRNA水平上升。共构建4个以不同长度细胞周期素D2启动子片段驱动的报告基因质粒,分别命名为pGL3-1816CD2-LUC、pGL3-1358CD2-LUC、pGL3-720CD2-LUC和pGL3-524CD2-LUC。在TSA处理条件下,启动子活性的变化在-524 bp删切质粒时消失。结论 TSA条件下细胞周期素D2 mRNA水平上升是启动子依赖的;细胞周期素D2启动子的TSA效应区域在-720～-524 bp之间。
Objective To study the regulatory mechanism of cyclin D2 in the treatment of trichostatin A (TSA). Methods Cyclin D2 mRNA levels were detected by RT-PCR. The cyclin D2 promoter fragment was amplified by high-fidelity PCR using the genomic DNA of A549 as a template and cloned into the pGL3-BASIC vector. Based on this, the promoters of different lengths were amplified by high-fidelity PCR Fragment. The relative promoter activity was determined by the firefly enzyme reporter gene method. Results TSA treatment conditions, the cyclin D2 mRNA levels increased. A total of 4 reporter plasmids driven by D2 cyclin D2 promoter fragments of different lengths were constructed and named pGL3-1816CD2-LUC, pGL3-1358CD2-LUC, pGL3-720CD2-LUC and pGL3-524CD2-LUC, respectively. Under TSA treatment, changes in promoter activity disappeared at -524 bp deletion plasmid. Conclusions The TSA-dependent increase of cyclin D2 mRNA level is promoter-dependent. The TSA effect region of cyclin D2 promoter is between -720 bp and -524 bp.