Musashi2在肠肿瘤形成中的功能与作用

发布时间：2018-02-26 03:16

本文关键词： Msi2 肠干细胞 APC 结直肠癌　出处：《中国农业大学》2015年博士论文　论文类型：学位论文

【摘要】：结直肠癌是人类致死率较高的癌症之一,因此揭示肠道内肿瘤发生的分子机制具有重要的意义。Musashi (Msi)是一个在进化上十分保守的RNA结合蛋白,其主要结构和表达模式在线虫(C. elegans)、果蝇(Drosophila)、海鞘(Ciona intestinalis)和整个脊椎动物种群之中都很保守。在哺乳动物体内,Musashi有两个同源基因,分别是Musashi1(Msil)和Musashi2(Msi2)。Msi1和Msi2在蛋白序列以及构成RNA结合域的序列上有着高度的同源性。近年来的文献报道显示,Msi1和Msi2蛋白在神经干细胞和乳腺干细胞以及造血细胞内的表达量很高,并且检测到在与之对应的神经胶质瘤,乳腺癌细胞和血癌细胞中表达量显著升高。这说明Msi1和Msi2在对干细胞内稳态调控和肿瘤形成的过程中起到了重要的作用。然而,到目前为止对Msi2在肠干细胞以及肠道肿瘤发生过程中的功能知之甚少。经研究发现,Msi2/MSI2蛋白在小鼠小肠干细胞存在的隐窝内表达,并且在人多种消化道癌组织中表达量升高。本研究利用DOX (Doxycycline)诱导人源Msi2过表达的转基因小鼠TRE-Msi2,系统地探索了Msi2/MSI2在小鼠小肠上皮组织肿瘤形成中的功能和作用机制。Msi2过表达引起TRE-Msi2小鼠小肠上皮细胞增殖加速,细胞分化受阻,细胞凋亡加速,隐窝的高度以及密度相较于野生型小鼠显著增高及增大。这些表型与APC蛋白功能缺失后小鼠肠道表型相似。并且通过GSEA (Gene set enrichment analysis)的分析发现Msi2过表达后的基因表达特征与APC (Adenomatous Polyposis Coli)功能缺失相似。APC作为Wnt信号通路重要的抑制因子,其突变与结直肠癌的发生密切相关。在肠道肿瘤模型小鼠APCmin/+小鼠体内过表达Msi2后,导致形成的腺瘤数量增加。以上结果表明Msi2/MSI2在肿瘤形成过程中起到了重要的作用。然而,过表达Msi2后并未检测到Wnt信号通路的激活,也并未观察到APC在mRNA和蛋白水平上的改变,这一结果显示Msi2/MSI2独立于Wnt信号通路促进了小肠肿瘤的形成。为了揭示Msi2的分子作用机制,我们通过Clip-Seq技术对Msi2/MSI2靶标基因在全基因组水平进行了筛选,发现PTEN、lrig1、p21和bmpr1α等肿瘤抑制因子是Msi2/MSI2的靶标基因。结合GSEA的结果和生化结果,我们证实Msi2/MSI2通过结合于PTEN基因的3'UTR来抑制PTEN mRNA的翻译。PTEN蛋白水平下调激活了PI3K-Akt-mTORC1信号。综上所述,本研究发现了Msi2/MSI2通过抑制PTEN激活了mTOR信号通路,并获得了与APC功能缺失相似的小肠上皮细胞的表型及基因表达特征。本研究首次揭示出Msi2/MSI2作为APC下游的关键致癌基因,有效的调控了一条平行于Wnt信号通路的PTEN-PI3K-Akt-mTORC1信号通路来促进小肠肿瘤形成。
[Abstract]:Colorectal cancer is one of the most fatal cancers in humans, so it is important to reveal the molecular mechanism of intestinal tumorigenesis. Musashi Msi is an evolutionarily conserved RNA binding protein. Its main structure and expression patterns are conserved in C. eleansa, Drosophila melanogaster, Ciintestinal inalis. and the whole vertebrate population. Musashi has two homologous genes in mammals. Musashi 1 (Msill) and Musashi2(Msi2).Msi1 and Msi2 have high homology in protein sequence and RNA binding domain. In recent years, it has been reported that Msi1 and Msi2 proteins are highly expressed in neural stem cells, mammary stem cells and hematopoietic cells. The expression levels in the corresponding gliomas, breast cancer cells and blood cancer cells were significantly increased. This suggests that Msi1 and Msi2 play an important role in the regulation of stem cell homeostasis and tumorigenesis. So far little is known about the role of Msi2 in the development of intestinal stem cells and intestinal tumors. It was found that Msi2 / MSI2 protein was expressed in the crypt of murine small intestinal stem cells. In this study, we used DOX Doxycycline to induce human Msi2 overexpression in TRE-Msi2 transgenic mice, and systematically explored the function and mechanism of Msi2/MSI2 in tumorigenesis of mouse intestinal epithelial tissue. Overexpression of Msi2 induced proliferation of intestinal epithelial cells in TRE-Msi2 mice. Cell differentiation is blocked, cell apoptosis is accelerated, The hight and density of crypt were significantly higher and larger than those of wild type mice. These phenotypes were similar to those of mice with APC protein deficiency. The gene surface of Msi2 overexpression was found by GSEA gene set enrichment analysis. Similar to the functional deletion of APC Adenomatous Polyposis Coli, APC is an important inhibitor of Wnt signaling pathway. The mutation is closely related to the occurrence of colorectal cancer. Overexpression of Msi2 in intestinal tumor model mice results in an increase in the number of adenomas. These results suggest that Msi2/MSI2 plays an important role in tumorigenesis. After overexpression of Msi2, no activation of Wnt signaling pathway was detected, and no changes of APC in mRNA and protein levels were observed. The results showed that Msi2/MSI2 was independent of Wnt signaling pathway and promoted the formation of small intestinal tumors. In order to elucidate the molecular mechanism of Msi2, we screened Msi2/MSI2 target genes at the whole genome level by Clip-Seq technique. We found that tumor suppressor factors such as PTENNlrig1p21 and bmpr1 伪 are the target genes of Msi2/MSI2. Combined with the results of GSEA and biochemical results, We confirm that Msi2/MSI2 activates PI3K-Akt-mTORC1 signal by inhibiting the level of translation. PTEN protein of PTEN mRNA by binding to 3UTR of PTEN gene. In conclusion, we found that Msi2/MSI2 activates mTOR signaling pathway by inhibiting PTEN. The phenotypic and gene expression characteristics of small intestinal epithelial cells similar to the absence of APC function were obtained. This study revealed for the first time that Msi2/MSI2 is the key oncogene downstream of APC. An PTEN-PI3K-Akt-mTORC1 signaling pathway parallel to the Wnt signaling pathway is effectively regulated to promote small intestinal tumor formation.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.3

【共引文献】