干细胞维持多能性分子机制的研究和长非编码RNA调控肿瘤细胞代谢的研究

发布时间：2018-06-18 16:12

本文选题：March5 + 胚胎干细胞　；参考：《中国科学技术大学》2015年博士论文

【摘要】：胚胎干细胞的多能性受到了胞内信号网络的精确调控,它对于胚胎干细胞将来在科研和医疗领域上的应用是十分重要的,但是它的具体机制仍然不为人们所清楚。在我们的研究工作中,我们利用了一种经逆转录病毒改造而来的载体pDisrup8,它可以随机的插入宿主细胞的基因组,造成插入突变,从而筛选和发现对于小鼠胚胎干细胞维持多能性所需要的关键基因。通过这样的方法,我们发现了基因March5参与干细胞干性的维持,它是一个泛素化连接酶。在敲低了March5后,会导致小鼠胚胎干细胞多能性标记基因表达水平的降低,干细胞克隆形成的能力受到损害和体细胞重编程效率的下降。与此同时,我们发现March5受到Klf4的转录调控,并且可以催化Prkarla的63位赖氨酸连接的多聚泛素化。Prkarla是蛋白激酶A的一个组成部分,它可以介导Raf-1第259位丝氨酸的磷酸化进而抑制下游MEK/ERK信号通路的活性。根据以往的报道,对MEK/ERK和GSK3信号通路的双重抑制可以支持胚胎干细胞在无血清条件下的生长。我们发现,外源表达March5或Klf4可以部分替代这个双重抑制体系中的MEK抑制剂。总而言之,我们的研究揭示一条新的维持干性的从Klf4到March5再到ERK的信号通路。根据近期的一些研究结果我们知道,体细胞重编程的过程也伴随着细胞代谢状态的重塑。苏氨酸脱氢酶(TDH)是苏氨酸代谢途径中最重要的酶之一,而且它仅在小鼠胚胎干细胞中特异性表达。然而,TDH介导的苏氨酸代谢是否会在体细胞重编程的过程中发挥作用仍然需要进一步的研究。我们的研究表明,TDH确实有这样的功能。敲低TDH会削弱重编程的诱导效率而过表达TDH会提高效率。同时,TDH受到microRNA-9的调控,而后者也会抑制重编程效率。另外,蛋白质精氨酸转移酶5(PRMT5)可以与TDH相互结合并使其第180位精氨酸发生甲基化。TDH的酶活性也因此受到PRMT5的调节,但这种调节通过PRMT5甲基转移酶活性依赖或不依赖两种方式进行的。重编程的诱导效率也会随着加入TDH的基础上再加入PRMT5而进一步提高。这些数据说明TDH及其下游的苏氨酸代谢参与到了体细胞重编程当中去,并且在转录后和翻译后两个水平上受到了多种调控。肿瘤细胞的重要特征之一就是Warburg效应,但其具体机制仍未完全清楚,而且长非编码RNA是否参与其中并发挥怎样的功能也不清楚。这里我们发现了一种受到c-myc调控的长非编码RNA (IDH1-AS1),其通过与异柠檬酸脱氢酶1(IDH1)结合,进而稳定其二聚体的形成,来维持其酶的活性,最终影响到肿瘤细胞HIF1A的水平而抑制Warburg效应。在小鼠的异种移植物模型中,敲低IDH1-AS1可以促进肿瘤生长。总之,这些数据表明IDH1-AS1是影响肿瘤Warburg效应的一个重要因子,可以作为肿瘤治疗的潜在靶点。
[Abstract]:The pluripotency of embryonic stem cells is precisely regulated by the intracellular signaling network, which is very important for the future application of embryonic stem cells in the field of scientific research and medicine, but its specific mechanism is still unclear. In our work, we used a retrovirus-modified vector, pDisrup8, that randomly inserted the genome of host cells, causing insertion mutations. The key genes needed to maintain pluripotency for mouse embryonic stem cells were screened and identified. In this way, we found that the gene March5 is involved in the maintenance of stem cell dryness, which is a ubiquitin ligase. After knocking down March5, the expression level of pluripotent marker genes of mouse embryonic stem cells was decreased, the ability of stem cell clone formation was impaired and the efficiency of somatic cell reprogramming was decreased. At the same time, we found that March5 is regulated by Klf4 transcription and catalyzes the polyubiquitin of 63 lysine ligation of Prkarla. Prkarla is a component of protein kinase A. It mediates the phosphorylation of serine at the 259th position of Raf-1 and inhibits the activity of the downstream MEK / ERK signaling pathway. According to previous reports, the dual inhibition of MEK / ERK and GSK3 signaling pathways can support the growth of embryonic stem cells in serum-free condition. We found that exogenous expression of March5 or Klf4 could partially replace MEK inhibitors in this dual inhibition system. All in all, our research reveals a new signal pathway that maintains dryness from Klf4 to March5 to ERK. According to some recent studies, somatic reprogramming is accompanied by remodeling of cell metabolic state. Threonine dehydrogenase (TDH) is one of the most important enzymes in threonine metabolism, and it is only specifically expressed in mouse embryonic stem cells. However, whether TDH mediated threonine metabolism may play a role in somatic reprogramming needs further study. Our research shows that TDH does have this function. Tapping down TDH weakens the induction efficiency of reprogramming, and over-expressing TDH improves efficiency. TDH is also regulated by microRNA-9, which also inhibits reprogramming efficiency. In addition, the protein arginine transferase 5 (PRMT5) can interact with TDH and make the enzyme activity of arginine methylation. TDH is regulated by PRMT5. But this regulation is done by PRMT5 methyltransferase activity dependent or independent. The induction efficiency of reprogramming will be further improved with the addition of PRMT5 on the basis of TDH. These data suggest that TDH and its downstream threonine metabolism are involved in somatic reprogramming and are regulated at both post-transcriptional and post-translational levels. Warburg effect is one of the important characteristics of tumor cells, but the mechanism of Warburg effect is not completely clear, and it is not clear whether long non-coding RNA participates in it and how it functions. Here we found a long non-coding RNAi IDH1-AS1G regulated by c-myc, which can stabilize the formation of dimer by binding with isocitrate dehydrogenase 1 and IDH1 to maintain its enzyme activity, and ultimately affect the level of HIF1A and inhibit Warburg effect. In mice xenografts, knocking down IDH 1-as 1 can promote tumor growth. All in all, these data suggest that IDH1-AS1 is an important factor affecting the Warburg effect and can be used as a potential target for tumor therapy.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R730.2

【共引文献】