Mechanism Research On Human Cdc25B Gene Transcriptional Regulation By IER5 In Irradiated HeLa Cells

Objective:Immediate early response 5,IER5,is a member of the slow-dynamic early response gene family,which is widely present in organs of the human body and is more prominent in the digestive,reproductive,and urinary tumor tissues than adjacent tissues.High expression of IER5 may be associated with poor prognosis of malignant tumors.In contrast,IER5 overexpression disrupts cell cycle checkpoints and produces radiation sensitivity in cells.However,the signaling pathways and mechanisms of IER5 in regulating the cell cycle remain unclear.This paper mainly focuses on how the IER5,which is up-regulated by radiation,carries out the regulation of periodic expression regulation.Then,to identify a key cell cycle regulatory protein,Cdc25B,affected by IER5.Using Cdc25B as a breakthrough to explore the role and specific mechanism of IER5 in the cell cycle regulation response of radiation injury.Methods:(1)4Gy y-ray irradiation was performed on HeLa cells with normal IER5 and IER5-silenced HeLa cells,and total RNA and total protein were collected at different time points after irradiation.qPCR and Western Blotting experiments were performed to detect how IER5 and Cdc25B mRNA and protein were detected,and the expression relationship between the two after irradiation was analyzed.(2)The Cdc25B gene promoter dual luciferase recombinant plasmid was constructed to investigate the effect of irradiation on the transcriptional activity of Cdc25B,and the promoter region in which the reaction element affected by radiation was present was found.Bioinformatics analysis was performed on this area,and overlap extension techniques were used to perform specific site mutations.The role of these specific sites in the regulation of radiation-mediated Cdc25B is further clarified.(3)In the 293T cells,the Cdc25B gene promoter dual luciferase recombinant plasmid and the IER5 expression plasmid were used to detect whether IER5 interacts with the Cdc25B promoter.(4)In HeLa cells,transiently transfect the point mutation plasmid and knock down IER5 with siRNA to see if IER5 can participate in radiation-mediated regulation of Cdc25B expression through a specific site.(5)The enrichment efficiency of key transcription factors of IER5,Spl,Sp3 and NF-YB in Cdc25B promoter region after irradiation was observed by ChIP-PCR.In HeLa cells,knock down IER5 with siRNA to see if IER5 knockdown affects radiation-mediated changes in several key transcription factors,Cdc25B.(6)Through qPCR and Western Blotting experiments to see whether the IER5 and NF-YB knockdown can rebound the Cdc25B down-regulation caused by NF-YB knockdown.Results:(1)HeLa cells and silenced-IER5 HeLa cells were exposed to ?-rays,and the expression changes between IER5 and Cdc25B were opposite.However,the percentage of Cdc25B in HeLa cells silenced by IER5 was significantly decreased at 8h(0.225 times vs 0.662 times,P<0.01).Therefore,it is believed that the up-regulation of IER5 expression by radiation has a regulatory effect on the down-regulation of Cdc25B expression induced by radiation.(2)Using the successfully constructed Cdc25B gene promoter dual luciferase recombinant plasmid,the Cdc25B promoter activity was significantly decreased after irradiation(P<0.001),and the radiation-affected response element was present in the-160?-53 region of the Cdc25B promoter.(3)IER5 expression can significantly reduce the transcriptional activity of Cdc25B(P<0.001),and the response elements affected by IER5 expression also exist in the-160?-53 region.Overexpression of IER5 reduces the activity of the Cdc25B gene promoter through the NF-YB binding site.(4)IER5 participates in radiation-mediated regulation of Cdc25B expression through the NF-YB binding site.(5)It was shown by ChIP-PCR gel electrophoresis that the IER5 protein was bound to the Cdc25B promoter.Compared with no irradiation,the enrichment efficiency of Spl,Sp3 and NF-YB decreased significantly after irradiation(P<0.01),but the enrichment efficiency of IER5 increased after irradiation(P<0.01).As a known coactivator of NF-YB interaction,p300 also released significantly after irradiation(P<0.01).After IER5 knocked down,the radiation-induced NF-YB binding release was raised.(6)By qPCR and Western Blotting experiments,the knockdown of IER5 and NF-YB could significantly reduce the down-regulation of Cdc25B caused by NF-YB knockdown.Conclusions:Irradiation causes up-regulation of IER5 expression in HeLa cells,which is regulated by transcriptional repression by binding more IER5 to NF-YB from the Cdc25B promoter region.