SAMD11信号通路及其在糖尿病视网膜病变中功能的研究

We found SAMD11 was the transcriptional co-activator of EYA. In the process of cloning full coding regions of SAMD11, we identified 45 SAMD11 alternative splice (AS) variants. Interestingly, 4 AS variants didn't obey the "GT-AG" rule. Among those 115 sequenced clones, ASV5 (20 out of 115) and ASV6 (26 out of 115) were the most dominant forms. ASV5 underwent AS events of E7c (139-186, E7) skipping and I6b (143-145, I6) retention; ASV6 yielded to I6b retention. Analysis of 98 ESTs related to human SAMD11 not only showed the wide expression of SAMD11 in different tissues, but also validated and enriched SAMD11 AS events that had been found. 96 ESTs related to mouse Samd11 indicated the existence of AS variants in mouse and the conservation of SAMD11 AS variants between human and mouse. Expression analysis by real-time PCR showed high expression of SAMD11 in 3 ocular cell lines (Y79, RPE, HCE) and 293T cells, while relatively low expression in HREC, HL-60 and SHI-1 cell lines. RT-PCR analysis revealed that the expression patterns of SAMD11 AS variants were similar in Y79 and 293T cells, while distinguished in other cells... Further analysis showed significantly increased expression of SAMD11 in diabetic retinopathy (DR) cell model, both in mRNA and protein levels, which indicated the important functions of SAMD11 in the pathogenesis of DR. To date, only one article focuses on mouse Samd11, there is no research on human SAMD11 function, let alone the roles of SAMD11 play in the pathogenesis of DR. Thus, to throw light on the pathology of DR, it is exigent to figure out the basic functions of SAMD11 at first...This study will focus on the interactions between SAMD11 and proteins, DNA, and analyzinge the functions and regulatory networks of SAMD11 with global view by the means of protein complex precipitation, ChIP-Seq, gene-expression profiling, etc. The analysis contents include proteins interact with SAMD11, signaling pathways downstream of SAMD11, promoters bound by SAMD11, regulation of SAMD11 expression, functions of SAMD11 AS variants, etc. The aim of this study is to reveal the functions of SAMD11 in the pathogenesis of DR, and to interfere, even block, development of DR by regulating the SAMD11 signaling pathway and interaction networks, and to provide molecular underpinnings for early diagnosis, treatment and intervention of DR.

申请者发现了SAMD11是EYA的协同转录因子；意外地了解到该基因存在45种选择性剪接变体（其中四种剪接变体不遵循传统的GT-AG规则），且这种特性在物种间相对保守；更令人兴奋的结果是：在高糖模型细胞中，SAMD11的mRNA和蛋白表达量都显著增加，提示SAMD11可能参与糖尿病视网膜病变（糖网）的发生发展。迄今为止，未见SAMD11功能的报道。.本课题拟用蛋白复合体沉淀、ChIP 测序（ChIP-Seq）和表达谱分析等方法，以SAMD11与蛋白、DNA相互作用为主要研究对象，全局性地分析SAMD11的功能及调控网络（顺式作用元件及反式作用因子、相互作用的蛋白、所调控的下游通路、所结合的启动子、选择性剪接变体的功能）；揭示其在糖网发生发展中的分子机制，发现糖网通路的新成员或阐明新的糖网通路，寻找治疗糖网的新靶点，为可能的早期诊断、治疗或干预方案提供分子依据。

了解转录因子SAMD11的基本特性。鼠Samd11基因充当转录抑制因子，主要表达在发育中的视网膜和成年小鼠的松果体中，其表达受到Crx的调控，Samd11基因可能参与小鼠视网膜分化发育过程，但是对人SAMD11基因尚无研究。从SAMD11基因剪接变体的鉴定、表达分析、转录调控等几个方面对SAMD11基因基本特性进行了研究。本研究通过对SAMD11基因基本特性的分析，发现人SAMD11基因功能可能不同于鼠Samd11基因，其可能不仅在眼组织发挥功能，还参与其他组织的生理进程。本研究丰富了人们对SAMD11基因的认识，为深入研究SAMD11功能提供基础。.SAMD11可能相关的转录因子（Notch信号通路）,是细胞分化生长的关键因子之一。分析了NOTCH信号通路的各个组分，发现其在RPE细胞中的表达水平各不相同。其中配体JAG1及转录激活子MAML1，MAML2的表达较其他组分高。抑制Notch通路能阻碍RPE细胞的增殖和迁移。流式细胞术检测表明Notch通路抑制前后RPE细胞的凋亡未有明显的变化，但对RPE细胞的周期影响较大，即抑制Notch通路后，S期及G2/M期的细胞明显减少，而G0/G1期细胞明显增多。NOTCH1的表达被抑制后影响Notch通路下游目的基因的表达。与SAMD11可能相关的转录因子等及其在眼科疾病中的可能作用。这部分的结果不仅丰富了人们对SAMD11可能相关的转录因子等在RPE和LEC细胞的功能的认识，还为RPE和LEC相关眼科疾病的研究和治疗提供了相关的理论基础。