Oct4异构体在干细胞和肿瘤细胞中的表达调控和作用机制

Oct4 is a crucial transcription factor in maintaining the pluripotency and self-renewal of embryonic stem (ES) cells. Human Oct4 gene can generate several transcript variants and protein isoforms (Oct4A, Oct4B-265, Oct4B-190 and Oct4B-164) by alternative splicing or alternative translation initiation. In embryonic stem cells, the expression and function of Oct4A have been studied extensively. However, the expression and function of other Oct4 isoforms are still under investigation, and there is limited information so far about Oct4 in tumour cells, including cancer stem cells. In our previous work, we found that human Oct4B mRNA could generate three protein isoforms through alternative translation initiation: the 265-amino-acid protein isoform Oct4B-265, the 190-amino-acid protein isoform Oct4B-190 and the 164-amino-acid protein isoform Oct4B-164; Oct4B-190 was found to be translated using an internal ribosome entry site (IRES)-mediated mechanism, up-regulated and antagonize cell apoptosis under stress. We also confirmed that Oct4B-265 was an isoform related to genotoxic stress in embryonic stem cells and embryonic carcinoma cells. In this study, we will further investigate the expression and function mechanism of each isoforms of Oct4 in stem cells and tumour cells. We will identify the cis-acting elements and trans-acting factors that regulate alternative splicing or alternative translation initiation of Oct4 isoforms, analyze their interaction and elucidate the regulation mechanism of Oct4 expression. We will further investigate the upstream and downstream regulatory factors of Oct4, determinate the signal transduction pathway that Oct4 isoforms participate in, and explore the biological function of Oct4 isoforms in stem cells and tumour cells. This work will give an insight into the complex expression patterns of Oct4 isoforms and help us to understand comprehensive biological functions of Oct4 gene in stem cells and tumour cells.

已发现维持干细胞多能性的关键基因Oct4可通过选择性剪接和翻译起始调控产生数种转录异构体和蛋白异构体。人们对胚胎干细胞中Oct4基因的表达和功能虽有较深入的研究，但对各种异构体的表达调控和相互作用机制还缺乏系统性了解，对肿瘤细胞包括肿瘤干细胞中Oct4的表达、调控和生物学功能还了解得很少。本课题拟在已有的工作基础上研究Oct4异构体在干细胞和肿瘤细胞中表达细节，分析和确定调控选择性剪接和翻译起始的顺序元件及反式作用因子，从调控元件与蛋白因子的相互作用中探索各异构体的表达调控机制，寻找Oct4的上游和下游调控因子及参与的信号转导通路，探索Oct4异构体在干细胞和肿瘤细胞中表达的变化规律、内在关系及其相互作用机制。通过本课题的研究将有助于我们鉴别Oct4不同蛋白异构体的表达、功能和调节机制的差异，系统理解Oct4异构体在维持干细胞的多能性和在肿瘤发生发展过程中的作用。

Oct4是维持胚胎干细胞多能性和自我更新相关的最重要的转录因子，同时也是体外诱导iPS的关键基因,已发现Oct4 可通过选择性剪接和翻译起始调控产生数种转录异构体和蛋白异构体,人们对胚胎干细胞中Oct4 基因的表达和功能虽有较深入的研究，但对各种异构体的表达调控和相互作用机制及其生物学功能还了解得很少。本课题拟在已有的工作基础上研究Oct4 异构体在干细胞和肿瘤细胞中表达细节，分析和确定调控选择性剪接和翻译起始的顺序元件及反式作用因子，探索Oct4 异构体在干细胞和肿瘤细胞中表达的变化规律、内在关系及其相互作用机制。.通过Western bloting，在胚胎干细胞和畸胎瘤细胞中检测到一种比Oct4A小一些的蛋白异构体。将Oct4A全长cDNA克隆到哺乳动物细胞表达载体pcDNA3.1上，转染到HEK293T细胞中进行表达，Western bloting检测，除了Oct4A蛋白异构体外，也检测到此较小的蛋白异构体；定点突变表达载体上的Oct4A全长cDNA的密码子，发现309位ATG突变成AGT后，此小蛋白异构体消失，推断此ATG为其起始密码子，此异构体由288个氨基酸组成，命名为Oct4-288，，比360个氨基酸组成的Oct4A蛋白异构体少了N端72氨基酸。. 突变Oct4A蛋白异构体的起始密码子ATG（74位）成AGT后会从紧邻的下游某框内非ATG起始密码子CTG（86位）起始翻译，效率要明显比原起始密码子低，同时可明显增加Oct4-288表达；进一步突变86位的CTG成CAG 后，仅检测到Oct4-288表达，且表达量显著增加，推断Oct4-288是通过漏过扫描（leaky scanning）的原理进行翻译。.通过本课题的研究将有助于我们鉴别Oct4 不同蛋白异构体的表达、功能和调节机制的差异，系统理解Oct4异构体在维持干细胞的多能性和在肿瘤发生发展过程中的作用。