探讨多梳家族蛋白PHC1敲降后对小鼠胚胎干细胞(mESCs)多能干性的影响,并探索其与多能干性基因nanog的转录调控机制。对mESCs细胞系进行PHC1蛋白沉默处理并设置阴性对照组(control组)和空白对照组(Empty vector组),采用qRT-PCR、Western blot、免疫荧光染色检测PHC1的表达量;荧光素酶报告基因技术(Luciferase Reporter)检测PHC1与nanog reporter之间的转录活性。结果提示:实验组mESCs呈现分化状态,多能干性基因nanog、oct4、sox2的表达下调,且nanog下调最显著;荧光素酶报告基因结果提示PHC1和nanog可能会形成复合物参与nanog的转录调控( P <0.05)。实验表明,敲降PHC1基因后可能通过影响nanog转录调控,从而参与核心转录调控“铁三角”(nanog、oct4、sox2)之间的相互转录调控机制,导致mESCs出现分化,进而参与mESCs多能干性的维持。
This study is aimed to investigate the effect of PHC1 gene knockdown to the pluripotency of mouse embryonic stem cells (mESCs), and to explore its transcriptional mechanism with multipotent gene nanog.The PHC1 gene of mESCs were silenced and empty vector control group was set up. The expression of PHC1 was detected by qRT-PCR, Western-Blot and immunofluorescence staining. The transcriptional activity between PHC1 and nanog reporter was detected by Luciferase Reporter. The results showed that the expression of multipotent genes nanog, oct4 and sox2 was down- regulated in the experimental group, especially,the down-regulation of nanog was the most significant. The results of luciferase reporter gene suggested that PHC1 and nanog might form a complex to participate in the transcriptional regulation of nanog reporter ( P <0.05).Therefore, knocking down PHC1 gene may affect the transcriptional regulation of nanog, and participate in the core transcriptional regulation of “iron triangle”(nanog, oct4, sox2), leading to the differentiation of mESCs and consequently participating in the maintenance of pluripotency on mESCs.
Journal of Medical Sciencein Central Sauh China