辐射介导下HeLa细胞中Cdc25B与IER5蛋白表达的关系

目的：探索辐射介导下HeLa细胞中Cdc25B与IER5蛋白表达的关系。方法：用4 Gy γ射线分别照射正常HeLa细胞和IER5-siRNA-HeLa细胞，收集照射后0、0.5、2、4、8、12、16、24、36 h的细胞样本，并设未照射细胞为对照组。采用qPCR法检测照射后Cdc25B mRNA的表达；Western blot检测细胞内IER5和Cdc25B蛋白的表达；流式细胞术检测照射后G2期细胞周期阻滞情况。结果：qPCR结果显示，照射后0.5~24 h内，与对照组相比，正常HeLa细胞内Cdc25B mRNA在0.5 h先下降2 h再上升之后再下降，差异均有统计学意义（P均 < 0.05）；在IER5-siRNA-HeLa细胞内Cdc25B mRNA在0.5 h先下降后再上升，差异均有统计学意义（P均 < 0.05，2 h组除外）。Western blot结果显示，照射后0.5~24 h内，与对照组相比，正常HeLa细胞内Cdc25B蛋白从2 h开始上升8 h开始下降，差异有统计学意义（P均 < 0.01）；IER5-siRNA-HeLa细胞内Cdc25B蛋白从0.5 h开始下降8 h开始逐渐上升，差异有统计学意义（P均 < 0.01，24 h除外）。照射后0~36 h内，与对照组相比，正常HeLa细胞与IER5-siRNA-HeLa细胞的IER5蛋白表达水平分别在照后8 h与0 h后开始出现明显上升，差异均具有统计学意义（P均 < 0.05）；而Cdc25B蛋白表达水分别在照射后8 h与0 h后开始出现下降，差异均具有统计学意义（P均 < 0.05），正常HeLa细胞与IER5-siRNA-HeLa细胞中Cdc25B蛋白与IER5蛋白的表达分别从8 h与0 h后开始呈现负相关关系（r分别为-0.740和-0.669，P均 < 0.01），当两种细胞中Cdc25B蛋白表达量下降时，G2期细胞周期阻滞比例升高（P < 0.05）。结论：辐射介导下HeLa细胞中Cdc25B蛋白表达变化是由Cdc25B mRNA变化引起的，并且Cdc25B与IER5蛋白表达呈负相关关系。

Expression of Cdc25B and IER5 in irradiated HeLa cells

OBJECTIVE:The purpose of this study was to investigate the relationship between the expression of Cdc25B and IER5 proteins in irradiated HeLa cells.METHODS:HeLa cells and IER5-knockdown Hela cells were irradiated with 4 Gy of γ rays and were harvested at 0,0.5,2,4,8,12,16,24,and 36 h after irradiation.Expression of IER5 and Cdc25B proteins was examined by Western blot.G2 cell cycle arrest was examined by flow cytometry.Expression level of Cdc25B mRNA was examined by real-time quantitative PCR.Unexposed cells were used as controls.RESULTS:The qPCR results show that within 0.5-24 h after irradiation of the normal HeLa cells the Cdc25B mRNA was lower from 0.5 h the time-pointand then increased after 2 h,the difference was statistically significant (P＜0.05);In IER5 knockdown Hela cells,Cdc25B mRNA decreased first and then increased after 0.5 h,the difference was statistically significant (all P＜0.05,except 2 h).The Western blot results show that within 0.5-24 h after irradiation of normal HeLa cells,the Cdc25B protein began to rise from the 2 h time-point,then began to decrease at 8 h,the difference was statistically significant (all P＜0.05,except 0.5 h).In IER5 knockdown Hela cells,Cdc25B began to decrease from the 0.5 h time-point,then began to rise at 8 h gradually,the difference was statistically significant (all P ＜0.05,except 24 h).Compared with the control group,IER5 protein was upregulated in two cells types after their irradiation and during the timepoints mentioned above.After radiation,the level of IER5 in HeLa began to increase significantly from the 8 h time-point while IER5 knockdown Hela cells from the 0 h.The differences were statistically significant (all P＜0.05).The level of Cdc25B in HeLa began to decline significantly from 8 h,while IER5 knockdown Hela cells from 0 h.The differences were statistically significant (P＜0.05).The protein expressions of Cdc25B and IER5 were negatively correlated at the 8 h and 0 h ime-points of radiation (R1=-0.740,R2=-0.669,P＜0.01,respectively),while the expression of Cdc25B was decreased.The ratio of G2 cell cycle arrest was increased in the two cells lines (P＜0.05).CONCLUSION:Expression of Cdc25B protein in HeLa cells was induced by Cdc25B mRNA changes,and the expression of Cdc25B was negatively related with the expression of IER5 protein.