转录因子OCT4通过细胞周期蛋白调控细胞命运的机理研究

OCT4 is a transcription factor encoded by POU5F1 gene. It is mainly expressed in embryonic stem cells, germline stem cells and embryonal carcinoma cells, and plays an indispensable role in maintaining the pluripotency and self-renewal of these pluripotent stem cells. Compared with the pivotal role of OCT4 in maintaining self-renewal, the research about cell cycle control of pluripotent stem cells by OCT4 is just at its infancy, and the underlying mechanisms remain to be fully elucidated. Our DNA microarray analyses and transcription factor-promoter binding predictions have indicated that the expression levels of a series of cell cycle genes were correlated with that of OCT4 during the differentiation of embryonal carcinoma cells and de-differentiation of glioblastoma cells. Building upon these preliminary findings, in the present study, we aim at systemically investigating the roles of OCT4 in regulating cell cycle proteins and profiling their dynamic changes during cell differentiation. We will then further explore the possibility of controlling the self-renewal of pluripotent stem cells and cancer stem cell by manipulating the expression of OCT4-regulated cell cycle proteins. Our ultimate goal is to elucidate the mechanisms by which OCT4 and cell cycle proteins work together to regulate cell fate determination.

OCT4是由POU5F1基因编码的转录因子，主要在胚胎干细胞、生殖干细胞和胚胎癌细胞中表达，对维持这些干细胞的多能性和自我更新具有无可替代的作用。相对于OCT4在自我更新中的关键作用，OCT4在干细胞细胞周期调控中的研究才刚起步，许多具体机制尚有待深入研究。我们通过基因芯片分析和转录因子-启动子结合预测等手段发现，在胚胎癌细胞分化和脑胶质瘤细胞去分化过程中有一系列细胞周期基因的表达与OCT4水平相关，并且是OCT4潜在的靶基因。本项目拟在前期工作基础上，系统分析OCT4对细胞周期蛋白的调控作用，揭示细胞周期蛋白在细胞分化过程中随OCT4蛋白差异表达而改变的动态过程；进一步探索通过改变OCT4相关细胞周期蛋白直接调控多能干细胞和肿瘤干细胞自我更新与定向分化等过程的可能性；最终阐释OCT4与细胞周期蛋白共同决定细胞命运的机制。

本项目旨在系统分析OCT4对细胞周期蛋白的调控作用，揭示细胞周期蛋白在细胞分化过程中随OCT4蛋白差异表达而改变的动态过程；进一步探索通过改变OCT4相关细胞周期蛋白直接调控多能干细胞和肿瘤干细胞自我更新与定向分化等过程的可能性；最终阐释OCT4与细胞周期蛋白共同决定细胞命运的机制。我们通过3年的努力，已基本完成了上述研究目标，主要取得了下列进展：.1. 构建了能指示成体肿瘤细胞中OCT4含量的GFP报告基因细胞株，找到受OCT4直接调控的周期相关蛋白SKP2,CDC45,WEE,MYT1,APC,CCND1,CCNE1,CCNA2,CCNB1,CDK4,CDK6等。.2.从肝癌和癌旁组织样本差异基因分析中发现OCT4能与两个细胞周期蛋白启动子区结合，直接调控其表达。.3.运用CRISPR/Cas9技术首次证实脑胶质瘤等分化的肿瘤细胞系中的OCT4A蛋白的差异表达会影响细胞周期蛋白的表达。.4.通过药物改变OCT4蛋白的翻译后修饰，从而直接调控的细胞周期蛋白表达，检测细胞周期蛋白调控肿瘤干细胞的自我更新过程的可能性。.5.提出OCT4、SOX2等干性基因在多能干细胞中通过调控G1期的R节点以及G2/M转变调控细胞周期并影响干细胞自我更新的模型。.我们在本项目资助下，迄今已发表4篇标注本项目的SCI论文。其中3篇论文影响影子>5。培养博士研究生3名，硕士研究生1名。项目组成员在国内外专业会议上报告了1次相关学术成果。.