ID4基因启动子分析、基因功能及在白血病细胞表达的初步研究
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摘要
本课题组通过生物信息学分析发现,ID4基因与白血病发生密切相关,是明确的抗白血病基因。以上述工作为基础进行了实验室研究。首先构建了6个5’端不同,3’端相同ID4-pGL3 Basic表达载体,转染MCF7细胞株,进行启动子活性检测,探讨ID4基因启动子及上游调控序列的特点,分析基因功能。下一步采用药物刺激实验,观察地塞米松、雌激素、二丁酰环磷腺苷三种生物信息学预测的药物对ID4基因启动子活性的调控作用。在上述工作的基础上,选择调控作用最强的雌激素与急性淋巴细胞白血病细胞系MOLT4细胞系共培养,靶向诱导ID4基因表达,同时观察细胞活力的变化。结果发现,ID4基因启动子核心调控区域在转录起始位点上游﹣272bp~﹣560bp,在﹣2200bp的区域内存在糖皮质激素受体元件、雌激素受体元件、cAmp受体元件三个顺式作用元件,能够上调ID4基因启动子的活性。其中,地塞米松受体顺式作用元件分布区域广泛,但作用缓和。而雌激素受体顺式作用元件分布区域虽然比较局限,但上调作用最明显。雌激素与MOLT4细胞共培养发现,雌激素可以靶向上调ID4基因表达,诱导细胞凋亡。
本课题首次分析了ID4基因的启动子活性,表达调控特点,并初步发现雌激素可以靶向诱导出MOLT4细胞(ID4基因表达阴性)ID4基因的表达,同时诱导细胞凋亡,具有良好的理论和实践意义。
Research Background: Adult acute lymphoblastic leukemia is a common type of leukemia, which has a poor prognosis. Although hematopoietic stem cell transplantation can cure some patients, most patients will eventually die because of recurrence. In recent years, the targeted cancer drugs obtain tremendous progress in the field of blood cancer, and bring hope to the treatment, but not in the acute lymphoblastic leukemia. Chinese scholars have found that, ID4 gene has anti-leukemia effect, and expression silence in acute lymphoblastic leukemia cells which may be related to abnormal methylation. It is a identified target genes related to leukemia. The full lenth of ID4 gene transcriptional copy has 1300 base of neucleotides, locates in 6P21~22, a inhibitor factor of helix loop helix transcription (bHLH). The coding region of ID4 protein HLH can adjust a variety of homologous or heterologous bHLH dimer, but it can not bind to a DNA common E-box to upregulate transcriptional function because of lack of DNA binding domain. but it can form heterodimers with the bHLH binding, prevente the combination of DNA and inactivate the bHLH transcription, stabilite basic transcriptional regulator protein dimer, regulate cell differentiation, proliferation and apoptosis.
Objectives:In order to explore the possible regulation mechnism of ID4 gene expression, study the novel gene’s function, and the affect of proliferation of acute lymphoblastic leukemia cells after targted upregulate ID4 gene.
Methods:①The present study first cloned promoter and sub-clone promoter molecules, then constructed sub-ID4 gene promoter-pGL3-Basic vectors. After that, Luciferase activity of all sub-clone promoter sequence were analysed. At last, the possible cis-acting elements were analysed by computer-aid system.②At the same time, the ID4 gene expression pattern was analyzed by the scanning of EST database, SAGE database, GeneNote database and 2D -PAGE database.③Based on these predictionresults, we feather study the promoter activity after drug stimulation. RealTime-PCR methods were used to detect ID4 gene expression after stimulation of Estrogen for 48h. CCK-8 methods detect cell activity at 24h, 48h and 72h Respectively.
Results:①ID4 gene promoter is a typical classⅡeukaryotic promoter with a TATA -box and several GC-box, which has a core positive regulation sequence from﹣272bp~﹣560bp and in the downstream of 5' flanking region of the TATA-box has two important region which regulate ID4 gene promoter activity. One of the boxes containing the E-box cis-acting element, which binds with the USF and activates ID4 gene promoter. The other is GA binding sequence, locates at downstream of the transcription start site whose mutation can upregulate transcriptional activity. a few putative positive cis-acting elements locating from﹣1bp~﹣1300bp, including Spl、C-Myb、abaA、C/EBPalpha、GR、ER and Zeste. While the existence of CCAAT-binding factor, GCF, WTl-KTS, HiNF-C and EGR2 acting elements likely to have a negative regulatory role.②Experiments show that the core positive regulation sequence locates from﹣270bp~﹣560bp, play a major role in expression regulation. Negative cis-acting elements locating from﹣560bp~﹣950bp,﹣1200bp~﹣2200bp, Positive cis-acting elements locating from﹣950bp~﹣1200bp. ID4 promoter activity increased after drugs (Glucocorticoid、Estrogen and cAmp) stimulation, of which the most strong is estrogen. Further verify the results of bioinformatics analysis.③Estrogen can stimulate the ID4 gene expression, inducate leukemia cell apoptosis.④ID4 gene is closely associated with leukemia, which may has somewhat function relation to leukemia and relaps.
Conclusions:①Analysed the features of ID4 gene promoter based on bioinformatics, and selected three drugs which targeted upregulate ID4 gene.②Laboratory studies had shown, ID4 gene has a core positive regulation sequence from﹣1bp~﹣560bp, and preliminary validated that there are glucocorticoid receptor element, estrogen receptor component, cAmp receptor component in the region, which can upregulate ID4 gene promoter activity Further illustrates the reliability and accuracy of bioinformatics.③Estrogen can stimulate the expression of ID4 gene and induce the apoptosis of MOLT4 cells.
本课题首次分析了ID4基因的启动子活性,表达调控特点,并初步发现雌激素可以靶向诱导出MOLT4细胞(ID4基因表达阴性)ID4基因的表达,同时诱导细胞凋亡,具有良好的理论和实践意义。
Research Background: Adult acute lymphoblastic leukemia is a common type of leukemia, which has a poor prognosis. Although hematopoietic stem cell transplantation can cure some patients, most patients will eventually die because of recurrence. In recent years, the targeted cancer drugs obtain tremendous progress in the field of blood cancer, and bring hope to the treatment, but not in the acute lymphoblastic leukemia. Chinese scholars have found that, ID4 gene has anti-leukemia effect, and expression silence in acute lymphoblastic leukemia cells which may be related to abnormal methylation. It is a identified target genes related to leukemia. The full lenth of ID4 gene transcriptional copy has 1300 base of neucleotides, locates in 6P21~22, a inhibitor factor of helix loop helix transcription (bHLH). The coding region of ID4 protein HLH can adjust a variety of homologous or heterologous bHLH dimer, but it can not bind to a DNA common E-box to upregulate transcriptional function because of lack of DNA binding domain. but it can form heterodimers with the bHLH binding, prevente the combination of DNA and inactivate the bHLH transcription, stabilite basic transcriptional regulator protein dimer, regulate cell differentiation, proliferation and apoptosis.
Objectives:In order to explore the possible regulation mechnism of ID4 gene expression, study the novel gene’s function, and the affect of proliferation of acute lymphoblastic leukemia cells after targted upregulate ID4 gene.
Methods:①The present study first cloned promoter and sub-clone promoter molecules, then constructed sub-ID4 gene promoter-pGL3-Basic vectors. After that, Luciferase activity of all sub-clone promoter sequence were analysed. At last, the possible cis-acting elements were analysed by computer-aid system.②At the same time, the ID4 gene expression pattern was analyzed by the scanning of EST database, SAGE database, GeneNote database and 2D -PAGE database.③Based on these predictionresults, we feather study the promoter activity after drug stimulation. RealTime-PCR methods were used to detect ID4 gene expression after stimulation of Estrogen for 48h. CCK-8 methods detect cell activity at 24h, 48h and 72h Respectively.
Results:①ID4 gene promoter is a typical classⅡeukaryotic promoter with a TATA -box and several GC-box, which has a core positive regulation sequence from﹣272bp~﹣560bp and in the downstream of 5' flanking region of the TATA-box has two important region which regulate ID4 gene promoter activity. One of the boxes containing the E-box cis-acting element, which binds with the USF and activates ID4 gene promoter. The other is GA binding sequence, locates at downstream of the transcription start site whose mutation can upregulate transcriptional activity. a few putative positive cis-acting elements locating from﹣1bp~﹣1300bp, including Spl、C-Myb、abaA、C/EBPalpha、GR、ER and Zeste. While the existence of CCAAT-binding factor, GCF, WTl-KTS, HiNF-C and EGR2 acting elements likely to have a negative regulatory role.②Experiments show that the core positive regulation sequence locates from﹣270bp~﹣560bp, play a major role in expression regulation. Negative cis-acting elements locating from﹣560bp~﹣950bp,﹣1200bp~﹣2200bp, Positive cis-acting elements locating from﹣950bp~﹣1200bp. ID4 promoter activity increased after drugs (Glucocorticoid、Estrogen and cAmp) stimulation, of which the most strong is estrogen. Further verify the results of bioinformatics analysis.③Estrogen can stimulate the ID4 gene expression, inducate leukemia cell apoptosis.④ID4 gene is closely associated with leukemia, which may has somewhat function relation to leukemia and relaps.
Conclusions:①Analysed the features of ID4 gene promoter based on bioinformatics, and selected three drugs which targeted upregulate ID4 gene.②Laboratory studies had shown, ID4 gene has a core positive regulation sequence from﹣1bp~﹣560bp, and preliminary validated that there are glucocorticoid receptor element, estrogen receptor component, cAmp receptor component in the region, which can upregulate ID4 gene promoter activity Further illustrates the reliability and accuracy of bioinformatics.③Estrogen can stimulate the expression of ID4 gene and induce the apoptosis of MOLT4 cells.
引文
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[2]赵俞,王全顺,李红华,等.四种恶性血液病细胞系的ID4基因启动子甲基化[J].中国实验血液学杂志, 2008, 16(6): 1418-1420.
[3]赵俞,王全顺,李红华,等. ID4基因启动子甲基化在急性髓系白血病完全缓解期检测中的意义[J].中国实验血液学杂志, 2008, 16(3): 476-478.
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