Study of Transcription Activity of X-Box Binding Protein 1 Gene in Human Different Cell Lines

人类不同类型细胞中X-盒结合蛋白1转录活性研究(英文)

Human X-box binding protein 1 （XBP1）, an important transcription factor, participates in many signal transduction processes. To further investigate the biological function of XBP1, sequences of XBP1 promoter and its two deletion mutants were first determined using bioinformatic analysis. The report vectors containing XBP1 promoter and its deletion mutants were then constructed, namely, p1-XBPlp, p2-XBPlp, and p3-XBPlp. Each reporter vector was separately transfected into HepG2, L02, K562, SMMC-7721, HSF, and Lipocyte lto Cell line using FuGENE 6 transfection reagents. The activity of chloramphenicol acetyltransferase （CAT） in each group of transfected cells was detected by ELISA assay, which in turn reflects the transcription activity of the XBP1 gene promoter. The activity involving p3-XBPlp was the highest in HepG2, which was 12.4-fold of that of pCAT3-Basic. The activities of p3-XBPlp in K562 and SMMC-7721 were the second and the third highest, which were 10.9-fold and 10.0-fold of that of the pCAT3-Basic, respectively. The CAT activity in L02 was lower than that in the above-mentioned abnormal cell, and no reporter activity was detected in HSF and Ito Cell. The XBP1 transcription and expression in K562, HepG2 and SMMC-7721 were found to be higher than that in L02, HSF and Ito cells, based on the results of real-time RT-PCR and Western blot. The XBP1 transcription and expression in L02, HSF was lower, whereas that in Ito cells was totally lacking. The result was similar to that of CAT-ELISA. Therefore, the XBP1 gene promoter can drive its downstream gene expression and its activity is cell line-dependent. The core sequence of XBP1 promoter was found between -227bp and 66bp sequence. This sequence was closely associated with the transcriptional activity of XBP1 promoter.

人类X-盒结合蛋白1(X-box binding protein1,XBP1)作为一种重要的转录因子,在细胞中涉及了广泛的信号调控过程。为进一步研究XBP1的生物学功能,首先利用生物信息学技术确定XBP1基因的启动子区域和2个缺失突变体的基因序列,聚合酶链反应扩增XBP1启动子和2个缺失突变体的基因序列,分别克隆至真核报告载体pCAT3-Basic中,构建3个报告载体p1-XBP1p、p2-XBP1p和p3-XBP1p,确定启动子活性最强的序列,并以该序列分别转染正常人肝细胞L02、人肝母细胞瘤细胞HepG2、人肝癌细胞SMMC-7721、人类红白血病细胞K562、人皮肤成纤维细胞HSF和人贮脂细胞Lipocyte Ito Cell 6种不同类型的细胞后(FuGENE 6 transfection reagents),CAT-ELISA方法检测氯霉素乙酰转移酶(CAT)在不同细胞系中的表达活性。每一组CAT结果反应了XBP1启动子转录活性的大小,其中p3-XBP1p在HepG2细胞中活性最强,是pCAT3-Basic的12.4倍,其次是K562和SMMC-7721,分别是10.9倍和10.0倍;在L02细胞中CAT酶活性低于上述3种异常细胞,在HSF和Ito细胞中CAT酶活性低或没有活性。运用适时荧光PCR方法和免疫印迹分别从mRNA水平和蛋白水平检测了XBP1在不同细胞中的表达情况,结果均显示XBP1在HepG2、K562和SMMC-7721细胞中的转录和表达强于L02、HSF和Ito细胞,在HSF细胞和L02细胞中转录和表达较低,在Ito细胞中几乎检测不到XBP1的表达,与CAT-ELISA检测结果一致。因此,XBP1启动子的转录活性在不同细胞中是具有差异的,而XBP1启动子转录活性的大小直接调控下游基因XBP1的表达,导致不同细胞中XBP1的表达丰度也不相同,XBP1启动子的转录活性和表达与细胞类型、细胞周期和组织特异性密切相关。本研究发现XBP1启动子的ATG上游-227bp～66bp区域与XBP1的转录活性密切相关,属于XBP1启动子的核心区域;进一步比较XBP1基因核心启动子区在不同细胞中转录活性的差别和XBP1基因表达丰度的差异,为揭示真核细胞中XBP1的转录调控机制奠定基础。