p53,miR-365在紫外线诱导小鼠胚胎成纤维细胞S期阻滞中作用

Effects of p53 and miR-365 on UV-induced S-phase arrest in mouse embryonic fibroblasts

目的探讨中波紫外线(UV-B)应激下p53调控细胞周期阻滞过程中miR-365的作用。方法用50 J/m2的UV-B分别照射p53正常表达的小鼠胚胎成纤维细胞(MEF)和p53低表达的MEF,分别在照前和照后不同时间点用流式细胞术检测细胞周期分布情况;用逆转录聚合酶链反应和蛋白质印迹(Western blot)分别检测p53在mRNA和蛋白表达水平的改变;用实时荧光定量聚合酶链反应检测miR-365的表达情况。结果 50 J/m2的UV-B照射后18 h,p53正常表达的MEF出现S期阻滞,并在照后24 h出现明显的凋亡峰,而在p53低表达的MEF中未观察到此变化;2种细胞其p53的mRNA表达水平在照后未见明显改变;p53正常表达的MEF的p53蛋白在照后1 h开始升高,至12 h达到峰值,24 h开始下降;而在p53低表达的MEF中p53蛋白未见明显改变;UV-B照射后p53正常表达的MEF中miR-365在照后4 h升高(P<0.05),并持续到照后8 h,照后12 h恢复到照前水平;而在p53低表达的MEF中miR-365无明显变化。结论UV-B照射可能通过转录后调控作用提高细胞p53蛋白水平进而发挥其诱导细胞S期阻滞的作用;miR-365参与了p53蛋白诱导S期阻滞的过程。

Objective To explore the role of miR-365 in p53-regulated cell cycle arrest induced by UV-B irradiation. Methods p53-normal-expressed MEF and p53-1ow-expressed MEF were exposed to UV-B irradiation at the dose of 50 J/m2. Flow eytometry was used to analyze the cell cycle distribution, reverse transcription PCR（RT-PCR） was used to examine the ex- pression of p53 mRNA, western blot was used to evaluate the level of p53 protein, and reahime quantitative PCR （ Reahime- qPCR） was used to detect the expression level of miR-365. Results The results of flow cytometry showed a pronounced S-phase arrest at the time of 18th hour and an apoptotic peak at 24th hour in p53-normal-expressed MEF after treated with 50 J/m2 UV-B, but not in p53-1ow-expressed MEF. There were no significant changes in p53 mRNA levels in both p53-normal-expressed MEF and p53-1ow-expressed MEF when they were treated with UV-B irradiation. The expression of p53 protein in p53-normal-ex- pressed MEF was increased 1 hour after UV-B exposure, which peaked at 12th hour, then decreased at 24th hour, but not chan- ges in the p53-1ow-expressed MEF. The expression of miR-365 in p53-nornlal-expressed MEF was increased after 4 hours, rea- ching maximun at 8th hour and returned to the basic level 12 hours after UV-B irradiation, but not changes in the p53-1ow-expre- sed MEF. Conclusion p53 was up-regulated in post-transcriptional level when exposed to UV-B irradiation and played an im- portant role in S-phase arrest ; miR-365 may join into the p53-regulated S-phase arrest when p53-normal-expressed MEF exposed to UV-B irradiation in this case.