The Interaction Of IER5With Cdc25B Promoter After Radiation In HepG2Cells

Objective: Hepatocellular carcinoma is a common malignant tumor,which is the fifth largest in the world and the third leading cause of cancerdeath. At present, the development of targeted therapy for liver cancer getslittle effect, this is largely due to cancer heterogeneity and the main signalingpathways have not yet been found. So the cell signaling pathways in theprocess of liver cancer occurrence and development are very important.Radioactive therapy for malignant tumor treatment is widely used. IER5, as amember of the IER family gene belongs to the slow dynamic genome, inducedexpression by ionizing radiation in various cells, play an important role inradiation induced cell death and cell cycle checkpoints. Cdc25phosphatase isa kind of double specific phosphatase that regulates the G2/M phase of thecell cycle. The abnormal expression of Cdc25B has been found widely invariety tumors, supporting that the Cdc25B expression is associated withtumorigenesis. The Cdc25B promoter analysis showed that the p53inhibitedthe Cdc25B expression dependent on binding the Cdc25B promoter. In recentyears, studies have shown that the IER5expression increased after exposure tothe radiation and participated in the expression of Cdc25B transcriptionalregulation. While the mechanism of the interaction of IER5with Cdc25Bis not clear, we decide to make the further study on it byRT-PCR,Semi-quantitative RT PCR,transient transfection assay,chromatinimmunoprecipitation technique and luciferase reporter gene assays.Methods:1Detect the IER5and Cdc25B mRNA levels in HepG2cellsat0,1,2,4,8,12h time point after Co60gamma-ray irradiation by Real-timequantitative polymerase chain reaction (PCR).2Inhibiting the IER5expression in HepG2cells by transient transfection assay, observe thevariation of the Cdc25B mRNA expression after Co60gamma-ray irradiation. We detect the Cdc25B mRNA levels in the peak hours after4Gy Co60gammairradiation by semi-quantitative polymerase chain reaction (PCR).3Detect thecombination of IER5and Cdc25B promoter after4Gy Co60irradiation at0,2,4,6,8h by chromatin immunoprecipitation technique.4Constructting Cdc25Bpromoter recombinant plasmids, the transcriptional activity variant wasdetected with aluciferase reporter in transient transfections of HepG2cellsafter4Gy Co60irradiation.Results:1After4Gy Co60irradiation, the IER5mRNA levels increased significantlycompared with control group in2h. The Cdc25B mRNA expressionsdecline in4h significantly. The opposite trends of the two genes suggestthat the increased amount of IER5expression may result in reducedCdc25B expression.2To prove that the Cdc25B mRNA decline after irradiation was caused byIER5, we observed Cdc25B mRNA levels after inhibitting IER5expression. The Cdc25B mRNA levels have no significantly declinecompared with the control group，thus supported that the IER5participatedin Cdc25B expression regulation.3Inhibitting IER5caused Cdc25B mRNA to accumulate, but its mechanismhas been unclear. We detected IER5and Cdc25B promoter combinationby chromatin immune coprecipitation. After4Gy Co60irradiation, in4hIER5and Cdc25B promoter combination increased significantly. Thisprove that IER5can combine with Cdc25B promoter, and the Cdc25Bpromoter is the target gene of IER5.4The Cdc25B promoter transcriptional activity was analyzed by Luciferasereport gene. Successfully constructed Cdc25B promoter recombinantplasmids,(-911,+105),(-575,+105),(-227,+105),(-122,+105) and (-51,+105)five pieces.The five different kinds of recombinant plasmids weretransfected in HepG2cells with the shIER5plasmids(experimental group)or mock-vehicle(control group).Compared with the control group, beforeirradiation the Cdc25B promoter transcriptional activity of the experimental group increased significantly which were transfected inrecombinant plasmids (-911,+105),(-575,+105),(-227,+105) and(-122,+105), on the other hand the cells transfected in plasmids(-51,+105)have no such changing. It indicated that the Cdc25B promotertranscriptional activity increased as well as the IER5being inhibitted, andthe acting sites may be in the (-122,-51) but not (-51,+105) region. Afterirradiation the transcriptional activity did not changed significantlybetween the two groups. The IER5expression induced by radiation lowerthe Cdc25B promoter transcriptional activity.Conclusion: Radiation induced IER5increased in HepG2cells, throughcombination with Cdc25B promoter, inhibit its transcriptional activity. Lowlevels of Cdc25B expression slow down the process of tumorigenesis. Lack ofIER5may promote the development of cancer. At the same time providing anew target therapy for liver cancer. At present, we have found that this part ofphenomenon, but also needs more work to explore.