Musashi在乳腺发育和肿瘤发生中的功能与分子作用机制
发布时间：2018-08-26 14:00
【摘要】：癌细胞转移是导致乳腺癌患者死亡的主要原因。上皮-间充质转化(EMT)是指细胞逐渐丧失正常上皮细胞特性,逐步获得间充质细胞特性的过程,在乳腺癌细胞转移中发挥关键作用。阐明EMT分子调控机制将有利于深入认识乳腺癌的转移机理,而且有助于开发新的药物设计靶点。Musashi (Msi)是进化上保守的RNA结合蛋白家族,在哺乳动物中包括两个同源蛋白：Msi1和Msi2。在机体正常发育过程中,Msi (Msi1和Msi2)主要在上皮组织表达,尤其在干/祖细胞中特异性高表达。有研究发现Msi在多种上皮来源的癌组织中高表达,并且在癌症发生过程中发挥着重要作用。然而,Msi在乳腺发育和乳腺癌中的功能,尤其是否参与调控EMT过程,目前仍未揭示清楚。本研究利用Msi过表达转基因小鼠、基因敲除小鼠和乳腺癌小鼠模型,结合体外细胞系培养,系统探究了Msi在乳腺发育和乳腺癌中的功能。本研究发现Msi1和Msi2的表达水平与乳腺癌亚型的侵袭转移能力密切相关。在转移能力较弱的腺腔样乳腺肿瘤中Msil和Msi2表现出更高的表达,相反在浸润性、转移能力较强的基底样乳腺肿瘤中表达水平均较低。进一步发现Msi与EMT分子标记基因呈负相关。在细胞水平,当敲减乳腺癌细胞中的Msil和Msi2基因时,细胞退出上皮状态转而进入间充质状态。为了验证Msi的体内生理功能,本研究首先制备了可诱导Msi2在乳腺上皮特异性过表达的转基因小鼠。过表达Msi2会导致乳腺导管延伸和分支受到显著抑制,腔上皮细胞数量增加,并且抑制EMT过程。相反,在Msil和Msi2双敲除小鼠中,乳腺上皮表现出促进EMT和肌上皮细胞数量增加。以上体外和体内实验结果表明,Msi在乳腺上皮中抑制EMT过程。为了进一步探究Msi在乳腺癌中的体内功能,本研究在MMTV-PyVT乳腺癌小鼠模型中过表达Msi2。发现Msi2过表达抑制乳腺肿瘤生长,同时通过抑制EMT过程显著抑制乳腺癌细胞的肺转移。因此,Msi是EMT的重要转录后调控因子,在乳腺癌转移中发挥重要作用。在分子作用机制上,本研究证实Jagged1 (Jag1)是Msi2的直接功能靶基因。利用分子生物学和生物化学实验证实了Msi2通过结合Jagl的3'UTR来抑制Jag1 mRNA的翻译。Jag1是Notch信号通路的配体,当Jagl表达水平升高时会激活Notch信号通路,进而促进EMT。当Jagl被Msi蛋白抑制后,细胞就被锁定在一种上皮状态。以上结果表明Msi2可以通过抑制Jag1-Notch信号通路来调控EMT过程。综上所述,本研究表明Msi在乳腺发育和乳腺癌中,通过靶向抑制Jagl的翻译和Notch信号通路的激活而抑制EMT过程和癌细胞转移。本研究首次提出了EMT的转录后调控机制,丰富了EMT的调控网络,为乳腺癌治疗药物的设计和策略的制定提供了科学依据。
[Abstract]:Cancer cell metastasis is the leading cause of death in breast cancer patients. Epithelial-mesenchymal transformation (EMT) is a process in which cells gradually lose normal epithelial cell characteristics and gradually acquire mesenchymal characteristics, which play a key role in breast cancer cell metastasis. Elucidating the molecular regulatory mechanism of EMT will be helpful to further understand the metastasis mechanism of breast cancer, and to develop a new drug design target. Musashi (Msi) is an evolutionarily conserved family of RNA binding proteins, including two homologous proteins: Msi1 and Msi2. in mammals. MSI (Msi1 and Msi2) are mainly expressed in epithelial tissues during normal development, especially in stem / progenitor cells. Some studies have found that Msi is highly expressed in cancer tissues from various epithelial sources and plays an important role in carcinogenesis. However, the role of MSI in breast development and breast cancer, especially whether or not it is involved in the regulation of EMT, remains unclear. In this study, Msi overexpression transgenic mice, gene knockout mice and breast cancer mice were used to investigate the function of Msi in breast development and breast cancer. In this study, we found that the expression of Msi1 and Msi2 was closely related to the invasion and metastasis ability of breast cancer subtypes. The expression of Msil and Msi2 was higher in adenoid breast tumors with weak metastatic ability, but lower in invasive and metastatic basal breast tumors. Further, negative correlation was found between Msi and EMT molecular marker gene. At the cellular level, when the Msil and Msi2 genes in breast cancer cells are knocked down, the cells withdraw from the epithelial state and enter the mesenchymal state. In order to verify the physiological function of Msi in vivo, transgenic mice which can induce the overexpression of Msi2 in breast epithelium were first prepared. Overexpression of Msi2 resulted in significant inhibition of breast ductal extension and branching, increased number of luminal epithelial cells, and inhibition of EMT process. In contrast, breast epithelium increased the number of EMT and myoepithelial cells in Msil and Msi2 double knockout mice. The results in vitro and in vivo indicate that Msi inhibits the EMT process in breast epithelium. In order to further explore the function of Msi in breast cancer, Msi2. was overexpressed in the mouse model of MMTV-PyVT breast cancer. It was found that the overexpression of Msi2 inhibited the growth of breast cancer and inhibited the lung metastasis of breast cancer cells by inhibiting the process of EMT. Therefore, Msi is an important posttranscriptional regulator of EMT and plays an important role in breast cancer metastasis. In molecular mechanism, Jagged1 (Jag1) is a direct functional target gene of Msi2. Molecular biology and biochemical experiments have confirmed that Msi2 inhibits the translation of Jag1 mRNA by binding 3'UTR of Jagl. Jag1 is the ligand of Notch signaling pathway, which activates Notch signaling pathway when Jagl expression level increases, thus promoting EMT.. When Jagl is inhibited by the Msi protein, the cells are locked in an epithelial state. These results suggest that Msi2 can regulate the EMT process by inhibiting the Jag1-Notch signaling pathway. In conclusion, this study suggests that Msi inhibits the EMT process and cancer cell metastasis by targeting the translation of Jagl and activation of the Notch signaling pathway in breast development and breast cancer. In this study, the posttranscriptional regulation mechanism of EMT is proposed for the first time, which enriches the regulatory network of EMT and provides a scientific basis for the design of breast cancer treatment drugs and the formulation of strategies.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9
，
本文编号：2205083
[Abstract]:Cancer cell metastasis is the leading cause of death in breast cancer patients. Epithelial-mesenchymal transformation (EMT) is a process in which cells gradually lose normal epithelial cell characteristics and gradually acquire mesenchymal characteristics, which play a key role in breast cancer cell metastasis. Elucidating the molecular regulatory mechanism of EMT will be helpful to further understand the metastasis mechanism of breast cancer, and to develop a new drug design target. Musashi (Msi) is an evolutionarily conserved family of RNA binding proteins, including two homologous proteins: Msi1 and Msi2. in mammals. MSI (Msi1 and Msi2) are mainly expressed in epithelial tissues during normal development, especially in stem / progenitor cells. Some studies have found that Msi is highly expressed in cancer tissues from various epithelial sources and plays an important role in carcinogenesis. However, the role of MSI in breast development and breast cancer, especially whether or not it is involved in the regulation of EMT, remains unclear. In this study, Msi overexpression transgenic mice, gene knockout mice and breast cancer mice were used to investigate the function of Msi in breast development and breast cancer. In this study, we found that the expression of Msi1 and Msi2 was closely related to the invasion and metastasis ability of breast cancer subtypes. The expression of Msil and Msi2 was higher in adenoid breast tumors with weak metastatic ability, but lower in invasive and metastatic basal breast tumors. Further, negative correlation was found between Msi and EMT molecular marker gene. At the cellular level, when the Msil and Msi2 genes in breast cancer cells are knocked down, the cells withdraw from the epithelial state and enter the mesenchymal state. In order to verify the physiological function of Msi in vivo, transgenic mice which can induce the overexpression of Msi2 in breast epithelium were first prepared. Overexpression of Msi2 resulted in significant inhibition of breast ductal extension and branching, increased number of luminal epithelial cells, and inhibition of EMT process. In contrast, breast epithelium increased the number of EMT and myoepithelial cells in Msil and Msi2 double knockout mice. The results in vitro and in vivo indicate that Msi inhibits the EMT process in breast epithelium. In order to further explore the function of Msi in breast cancer, Msi2. was overexpressed in the mouse model of MMTV-PyVT breast cancer. It was found that the overexpression of Msi2 inhibited the growth of breast cancer and inhibited the lung metastasis of breast cancer cells by inhibiting the process of EMT. Therefore, Msi is an important posttranscriptional regulator of EMT and plays an important role in breast cancer metastasis. In molecular mechanism, Jagged1 (Jag1) is a direct functional target gene of Msi2. Molecular biology and biochemical experiments have confirmed that Msi2 inhibits the translation of Jag1 mRNA by binding 3'UTR of Jagl. Jag1 is the ligand of Notch signaling pathway, which activates Notch signaling pathway when Jagl expression level increases, thus promoting EMT.. When Jagl is inhibited by the Msi protein, the cells are locked in an epithelial state. These results suggest that Msi2 can regulate the EMT process by inhibiting the Jag1-Notch signaling pathway. In conclusion, this study suggests that Msi inhibits the EMT process and cancer cell metastasis by targeting the translation of Jagl and activation of the Notch signaling pathway in breast development and breast cancer. In this study, the posttranscriptional regulation mechanism of EMT is proposed for the first time, which enriches the regulatory network of EMT and provides a scientific basis for the design of breast cancer treatment drugs and the formulation of strategies.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9
，
本文编号：2205083
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