A Primary Study Of ID4 Gene Promoter Analysis,Gene Function And Its Expression In Leukemia Cells

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:â‘ The 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.â‘¡At the same time, the ID4 gene expression pattern was analyzed by the scanning of EST database, SAGE database, GeneNote database and 2D -PAGE database.â‘¢Based 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:â‘ ID4 gene promoter is a typical classâ…¡eukaryotic promoter with a TATA -box and several GC-box, which has a core positive regulation sequence fromï¹£272bp~ï¹£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 fromï¹£1bp~ï¹£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.â‘¡Experiments show that the core positive regulation sequence locates fromï¹£270bp~ï¹£560bp, play a major role in expression regulation. Negative cis-acting elements locating fromï¹£560bp~ï¹£950bp,ï¹£1200bp~ï¹£2200bp, Positive cis-acting elements locating fromï¹£950bp~ï¹£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.â‘¢Estrogen can stimulate the ID4 gene expression, inducate leukemia cell apoptosis.â‘£ID4 gene is closely associated with leukemia, which may has somewhat function relation to leukemia and relaps.Conclusions:â‘ Analysed the features of ID4 gene promoter based on bioinformatics, and selected three drugs which targeted upregulate ID4 gene.â‘¡Laboratory studies had shown, ID4 gene has a core positive regulation sequence fromï¹£1bp~ï¹£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.â‘¢Estrogen can stimulate the expression of ID4 gene and induce the apoptosis of MOLT4 cells.