Study On The Relationship Between IER5 And Cdc25B Expression In HeLa Cells Mediated By Radiation

Objective:The purpose of this stduy is to explore the biological function of IER5 in HeLa cells after irradiation and the relationship between IER5 and Cdc25B expression.Methods:cell samples of 0,0.5,2,4,8,12,16,24,36,48h were collected from normal HeLa cells and siIER5-HeLa cells after 4 Gy gamma ray irradiation,and the unirradiated cells were set as the control group.The expression of IER5 and Cdc25B protein was detected by Western blot.The cell cycle arrest in G2 phase after irradiation was detected by flow cytometry.In addition,siNC and siIER5 were transfected to HeLa cells,IER5 overexpressed plasmids were transfected to siIER5-HeLa cells respectively,and the cells were irradiated with 2Gy and 4Gy gamma rays,and cell samples of 0.5,2,4,8,12,24h and control groups were collected after irradiation,and the intracellular G2/M phase arrest,the mRNA and protein levels of IER5 and Cdc25B was detected by flow cytometry,qPCR and Western blot.Through bioinformatics analysis and literature review,the regulatory factors that may be involved in the regulation of Cdc25B gene transcription by IER5 were selected.After interfering with the expression of IER5 in HeLa cells,the changes of transcriptional regulatory proteins were detected to preliminarily explore the possible mechanism of IER5 affecting Cdc25B transcription after radiation mediated.Results:1.The expression level of IER5 in HeLa cell group and siIER5-HeLa cell group was increased by 8h and Oh(*P<0.05),respectively,and the expression level of Cdc25B decreased at the corresponding time point after 4Gy irradiation(*P<0.05).In addition,G2/M phase arrest occurred in two groups of 8h cells.2.After 2Gy and 4Gy doses irradiation,the percentage of G2/M phase in HeLa cells transfected with siIER5 was lower than that transfected with siNC.At the same time,transfection with siIER5 resulted in the time delay in the up regulation of IER5 expression in HeLa cells and the change of the time point and expression of Cdc25B protein and mRNA.When IER5 is up-regulated(*_P<0.05),the expression of Cdc25B is down(*P<0.05).3.The proportion of siIER5-HeLa cells transfected with IER5 overexpressed plasmid after 2Gy and 4Gy dose irradiation was significantly higher than that in siIER5-HeLa cell group transfected with empty vector plasmid,and the duration of block duration was longer.Meanwhile,transfection with IER5 over expression plasmid leads to the up regulation of IER5 expression,and it also causes the down regulation of Cdc25B protein and mRNA level.4.Bioinformatics analysis showed that p300,p53 and NF-YB may be important regulatory proteins involved in IER5 mediated Cdc25B transcription.The changes of p300,p53 and NF-YB protein levels in HeLa cells before and after interference of IER5 were detected by different radiation doses.The p300 protein was greatly influenced by IER5,and the level of p300 protein decreased after the interference of IER5 expression(*P<0.05),while the expression of p53 was greatly influenced by the dose of radiation,and was less affected by IER5.However,the level of NF-YB protein was not affected by the expression of IER5,but only affected by the dose of radiation.Conclusions:1.There was a negative correlation between the expression of IER5 and Cdc25B protein in HeLa cells after 4Gy dose irradiation,the correlation coefficient was-0.686 and-0.633,and the change of IER5 expression could affect the cell cycle distribution,and the proportion of arresting cell cycle at G2/M phase increased when the IER5 expression increased.2.Transfection with siIER5 to HeLa cells leads to a decrease in IER5 expression,which can alleviate G2/M phase arrest induced by 2Gy and 4Gy radiation dose,and IER5 is the upstream regulation protein of Cdc25B.3.The up regulation of IER5 expression induced by transfection with IER5 over expression plasmids to siIER5-HeLa could lead to up regulation of G2/M block and prolongation of blocking time caused by 2Gy and 4Gy radiation doses,and IER5 was negative regulation of Cdc25B expression from transcriptional level.4.There is a possibility that IER5 affects the transcription of Cdc25B by regulating the level of p300 protein after irradiation,and the level of p53 protein is affected by IER5 and dose,and IER5 has no regulatory relationship to the transcriptional regulatory protein NF-YB of Cdc25B.