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目的分析人类转录因子X-盒结合蛋白1(X-box binding proteinⅠ,XBP1)基因启动子在不同细胞系中转录活性的差别,研究其转录调控机制。方法在对XBP1基因进行生物信息学分析的基础上,设计6种XBP1启动子缺失突变体,分别包括XBP1基因5′上游-1039~66bp、-859~66bp、-623~66bp、-351~66bp,-227~66bp、-227~-45bp,针对每一种缺失突变体构建相应的氯霉素乙酰转移酶(chloramphenicol acetyltansferase,CAT)报告基因载体,再分别瞬时转染K562、HepG2、NIH-3T3和L02细胞,应用报告基因CAT瞬时表达系统,检测XBP1每一种缺失突变体在K562、HepG2、NIH-3T3和L024种不同细胞中的活性差异,并进一步确定XBP1基因启动子的具体调节部位。结果成功获得XBP1基因启动子6种缺失突变体的正确克隆和6种相应的报告基因载体p1-XBP1p,p2-XBP1p,p3-XBP1p,p4-XBP1p,p5-XBP1p,p6-XBP1p,每一种报告基因转染K562、HepG2、NIH-3T3和L024种不同细胞系后,p4-XBP1p和p5-XBP1p在K562、HepG2细胞中的转录活性均高于其它报告载体,p5-XBP1p在HepG2细胞中转录活性最高;而6种报告基因载体在NIH-3T3小鼠成纤维细胞中不具有转录活性。结论XBP1基因的转录激活能力在不同细胞中有所差异,其表达活性具有一定的细胞类型特异性,与细胞类型和细胞状态密切相关;XBP1基因启动子的-227~66bp区域是该启动子的核心部位,而且核心启动子区包括ATG翻译起始密码子的下游部位,这一部位是转录活性的重要部位,一旦缺失,XBP1基因启动子会丧失活性。
OBJECTIVE: To analyze the difference of transcriptional activity of human X-box binding protein 1 (XBP1) gene promoter in different cell lines and study its transcriptional regulation mechanism. Methods Based on the bioinformatics analysis of XBP1 gene, six XBP1 promoter deletion mutants were designed, including -1039 ~ 66bp, -859 ~ 66bp, -623 ~ 66bp, -351 ~ 66bp , -227 ~ 66bp, -227 ~ -45bp, corresponding chloramphenicol acetyltansferase (CAT) reporter vector was constructed for each deletion mutant, and then transiently transfected into K562, HepG2, NIH-3T3 And L02 cells. The reporter gene CAT transient expression system was used to detect the differences in the activity of each of the XBP1 deletion mutants in K562, HepG2, NIH-3T3 and L024 different cells, and to further determine the specific regulatory site of the promoter of XBP1 gene. RESULTS: The correct clones of the six deletion mutants of XBP1 gene promoter and the six corresponding reporter vectors p1-XBP1p, p2-XBP1p, p3-XBP1p, p4-XBP1p, p5-XBP1p and p6-XBP1p were successfully obtained. The transcriptional activity of p4-XBP1p and p5-XBP1p in K562 and HepG2 cells transfected with reporter gene K562, HepG2, NIH-3T3 and L024 were higher than those of other reporter vectors and the transcription of p5-XBP1p in HepG2 cells While the six reporter vectors did not show transcriptional activity in NIH-3T3 mouse fibroblasts. Conclusion The transcriptional activation of XBP1 gene is different in different cells. The expression of XBP1 gene has certain cell type specificity and is closely related to cell type and cell status. The -227 ~ 66bp region of XBP1 gene promoter is Core region, and the core promoter region includes the ATG translation initiation codon downstream site, which is an important part of transcriptional activity. Once deleted, the XBP1 gene promoter will lose its activity.