Effect of CHD4 gene expression on proliferation and apoptosis of acute T lymphocytic leukemia cells and its promoter identification
Author:
Affiliation:
Fund Project:
摘要
|
图/表
|
访问统计
|
参考文献
|
相似文献
|
引证文献
|
资源附件
|
文章评论
摘要:
目的：探讨人染色质解旋酶DNA结合蛋白4（recombinant chromodomain helicase DNA binding protein 4，CHD4）基因表达对急性T细胞淋巴细胞白血病细胞增殖和凋亡的影响及其启动子鉴定。方法：使用siRNA-CHD4瞬时转染T淋巴细胞白血病细胞（Jurkat）敲低CHD4基因表达，实时荧光定量qRT-PCR和Western blot检测细胞转染后CHD4表达量，流式细胞术测定细胞凋亡率及细胞周期变化；CCK-8分析测定CHD4基因对Jurkat细胞增殖的影响。根据生物信息学分析，以Jurkat细胞提取的全基因组DNA为模板，PCR扩增CHD4基因候选启动子区2 091 bp片段，以pGL3-Basic为载体，克隆含有CHD4基因候选启动子区的序列，制备重组质粒，并且构建一系列含CHD4基因候选启动子5′侧翼区截短序列质粒。将含CHD4启动子序列的质粒及截短序列质粒转染至Jurkat细胞和人胚胎肾T细胞（HEK293T），双荧光素酶报告基因检测各片段的启动子活性，确定CHD4基因启动子最小活性区域，并通过生物信息学方法分析该区域转录因子结合位点，构建结合位点突变的质粒，通过双荧光素酶报告基因检测，分析结合位点对CHD4 转录的影响。结果：流式细胞检测结果发现，与对照组比较，CHD4抑制Jurkat细胞凋亡，转染siRNA-CHD4的Jurkat细胞G0/G1期比例显著升高，而S期比例下降（P＜0.01）；CCK-8检测CHD4基因对Jurkat细胞的增殖有促进作用（P＜0.05）；成功构建含有CHD4基因候选启动子序列的质粒及其截短序列质粒，与pGL3-Basic空载体相比，含有CHD4基因候选启动子序列的质粒活性明显增加（P＜0.05）。CHD4基因最小活性区域位于转录起始位点-233~-13 bp，其中包含NF-κB、MZF1等转录因子结合位点；NF-κB 对CHD4启动子活性具有正向调控作用。结论：CHD4基因对Jurkat细胞凋亡有抑制作用，并且促进其增殖。CHD4最小活性区域位于转录起始位点-233~-13 bp，转录因子NF-κB 对CHD4基因启动子活性具有正向调控作用。
Abstract:
Objective：This study aims to explore influences of recombinant chromodomain helicase DNA binding protein 4（CHD4）gene expression on proliferation and apoptosis of acute T lymphoblastic leukemia cells and to identify its promoter. Methods：siRNA-CHD4 was used to transiently transfect Jurkat cells to knock down the expression of CHD4. The qRT-PCR and Western blot were used to detect the expression of CHD4. The apoptosis rate and cell cycle were measured by flow cytometry. The effect of CHD4 gene on the proliferation of Jurkat cells was analyzed by CCK-8. According to bioinformatics analysis，a 2 091 bp fragment of CHD4 gene candidate promoter region was amplified by PCR using the whole genome DNA extracted from Jurkat cells as template. The sequence containing candidate promoter region of CHD4 gene was cloned with pGL3 basic as vector，and a series of plasmids containing truncated 5′ flanking region of CHD4 gene candidate promoter were constructed. The plasmids containing CHD4 promoter and truncated sequence were transfected into Jurkat and HEK293T cells. The promoter activity of each fragment was detected by double luciferase reporter gene，and the minimal active region of CHD4 gene promoter was determined. The effect of binding site on the transcription of CHD4 was analyzed by double luciferase reporter gene detection. Results：Flow cytometry showed that，compared with the control group，CHD4 inhibited the apoptosis of Jurkat cells，the Jurkat cells transfected siRNA-CHD4 were significantly increased in the G0/G1 phase and decreased in the S phase（P < 0.01）. CCK-8 essay identified that CHD4 gene promoted the proliferation of Jurkat cells（P < 0.05）. Plasmids containing CHD4 gene candidate promoter and truncated sequence plasmids were successfully constructed. Compared with empty vector，plasmids containing CHD4 gene candidate promoter sequences were significantly more active（P < 0.05）. The core promoter of CHD4 gene located in 233 bp to 13 bp relative to transcription start site，which contained transcription factor binding sites NF-κB and MZF1. NF-κB had a positive function to the CHD4 promoter activity. Conclusion：Our results suggested that CHD4 inhibited apoptosis and induced proliferation in Jurkat cells. The core promoter of CHD4 located in -233/-13 bp relative to TSS. NF-κB bound to the core promoter of CHD4 in vivo and it had positive function to the promoter activity of CHD4.
