miR-34a在辐射损伤时对细胞周期的调控作用

目的探讨60Coγ射线诱发大鼠肝脏BRL细胞辐射损伤时p53激活诱导的miR-34a改变对细胞周期的调控作用。方法以大鼠BRL肝细胞为研究对象,予4 Gy60Coγ射线照射后,分别培养4、12、24、48 h。采用流式细胞技术检测各组BRL细胞周期的变化,实时定量PCR检测各组细胞中miR-34a mRNA和c-myc mRNA表达水平的变化,Western blot方法检测各组细胞中p53蛋白和Myc蛋白的表达。结果 60Coγ射线4 Gy照射后4 h即出现G2期阻滞（P〈0.05）,至照射后24 h,G2期阻滞恢复;照射后12 h起,S期细胞减少（P〈0.05）,出现G1阻滞,48 h仍未恢复。照射后4 h p53蛋白和miR-34a mRNA表达增加（P〈0.05）,12 h均有下降（P〈0.05）,24 h和48 h已基本回至正常水平,而c-myc在照射后4 h开始呈持续降低趋势,48 h有所恢复。结论细胞在电离辐射引起DNA损伤后,早期即激活了p53蛋白,p53蛋白调节miR-34a表达,可能再经由miR-34a的靶基因c-myc介导,使细胞阻滞于G1和（或）G2期进行DNA修复。

Regulation of the Cell Cycle By miR-34a in the Radiation Damage Response

Objective To investigate the effects of the miR-34a activated by p53 on the BRL cell cycle when the BRL cell were induced damage by 60Co γ-ray.Methods The rat BRL cells were exposured to 4Gy 60Co γ-ray and there was no irradiation were taken as the control.After irradiation,the cells were respectively cultured for 4 h,12 h,24 h and 48 h.Flow cytometry was applied to detect the changes of the BRL cell cycle;Real-time PCR assay was applied to detect the expression of miR-34a and c-myc gene mRNA;p53 and c-myc protein were detected by Western blot assay.Results After 4Gy 60Co γ-ray irradiation,G2 arrest appeared at the 4th hour and restored at the 24th hour;the population of S cells decreased at the 4th hour and G1 arrest appeared,which proceeded to the 48th hour.The expressions of p53 protein and miR-34a mRNA increased at the 4th hour,decreased at the 12th hour and restored at the 24th hour after irradiation,the expression of c-myc decreased continuously in a time-dependent manner（the 4~24th hour） and restored to the level of the control at the 48th hour.Conclusions After DNA damage in cells is caused by ionizing radiation,p53 protein is activated in the early and the expression of miR-34a is induced which may mediate the target gene c-myc,resulting in the cells arresting in G1 or/and G2 phase to repare DNA.