转录因子ZBTB7B在小鼠乳腺发育过程中的功能及机制研究

The mammary gland (MG) develops through several distinct stages including embryonic, prepubertal, pubertal, pregnancy, lactation and involution. These processes require numerous signaling pathways that have distinct regulatory functions at different stages. Study of the key regulatory molecules and signaling pathways in MG development, is of great significance to understand the occurrence and development of breast disease and breast cancer. The mammary duct is composed of luminal epithelial cells which derived from mammary stem cells and basal cells rich in stem cells. Our previous studies found that the transcription factor ZBTB7B was specifically expressed in luminal epithelial cells in MG, and total knockout of Zbtb7b in mice led to a decrease of luminal cells and an increase of basal cells, suggesting that ZBTB7B may be involved in the regulation of differentiation of mammary epithelial cells. Meanwhile, we found that ZBTB7B was downregulated during EMT, the overexpression of ZBTB7B can inhibit TGF-β, SMAD3 or SNAIL induced EMT, and knockdown ZBTB7B would directly lead to EMT. These data demonstrated that ZBTB7B is a critical regulator of EMT. However, the molecular mechanism of ZBTB7B in regulating EMT and how its expression level changed during EMT is still unknown. EMT plays an important role in embryonic development of MG, differentiation of mammary epithelial cells and maintenance of mammary stem cells, but the mechanism has not been elucidated. We speculate that ZBTB7B may affect mammary gland development by regulating EMT. In this project, we will systematically study the role and regulation mechanism of ZBTB7B in the process of mammary gland development, in order to discover the new function of ZBTB7B and new molecular mechanism in affecting mammary gland development via regulating EMT.

乳腺发育包括胚胎期、青春期、成熟期、怀孕期、泌乳期和退化期。深入研究每一阶段的关键调控分子和信号通路，对了解乳腺疾病及乳腺癌的发生发展具有重要意义。乳腺导管由腔上皮细胞和富含干细胞的肌上皮细胞组成，前者由乳腺干细胞分化而来。我们的前期工作发现，在乳腺导管中，转录因子ZBTB7B特异表达于腔上皮细胞；全身敲除Zbtb7b导致腔上皮细胞比例降低，肌上皮细胞比例升高，提示ZBTB7B可能参与调控乳腺上皮细胞的分化。同时，我们发现ZBTB7B在EMT过程中表达下调；过表达ZBTB7B能抑制多种方式诱导的EMT，敲低则会导致EMT的发生。EMT在乳腺上皮细胞分化及乳腺干细胞的维持等过程中发挥着重要功能，但其机制并不清楚。我们推测ZBTB7B可能通过调控EMT进而影响乳腺的发育。本项目将系统的研究小鼠乳腺发育过程中ZBTB7B的作用及调控机制，以期发现ZBTB7B的新功能及其调控乳腺发育的分子机制。

转录因子ZBTB7B是决定细胞命运的关键调节因子，其参与了T细胞的分化、棕色脂肪发育和冷诱导的米色脂肪形成等多个生物学过程。除细胞命运调控外，ZBTB7B还参与了代谢调控。ZBTB7B通过直接调节胰岛素受体下游关键接头蛋白IRS-1的表达，调控胰岛素诱导的Akt-mTOR-SREBP通路，进而影响了哺乳期乳腺腺泡细胞中的脂质合成。然而，ZBTB7B是否具有其他的生理病理功能还有待探索。本实验室的前期研究发现，ZBTB7B在小鼠乳腺导管中特异表达于腔上皮（luminal）细胞中，提示ZBTB7B可能在小鼠乳腺发育过程中发挥功能。本研究发现，在乳腺细胞系MCF-10A中，ZBTB7B可能通过调控SLUG的表达来抑制上皮间质转化 (epithelial-mesenchymal transition, EMT) 的进程；进一步我们通过构建Zbtb7b基因乳腺腔上皮细胞特异性敲除小鼠，发现ZBTB7B的缺失可能通过影响EMT促进小鼠的退化期。在对ZBTB7B与小鼠乳腺发育的关系进行研究的同时，我们发现ZBTB7B在小鼠肝脏中表达较高，提示我们其在肝癌的发生发展中也有可能发挥作用。通过构建Zbtb7b基因肝脏特异性敲除小鼠，结合Akt/N-Ras诱导的肝癌小鼠模型，我们发现ZBTB7B能够直接调控c-Jun的表达，进而影响肝癌的起始，具有抑癌的功能。与ZBTB7B相似，次要类型IV型胶原蛋白α5链 (COL4A5) 在乳腺腔上皮细胞及管腔型乳腺癌细胞中高表达。我们利用自发乳腺癌MMTV-PyVT小鼠模型与Col4α5基因乳腺腔上皮细胞特异性敲除小鼠，发现COL4A5的缺失能够抑制乳腺原位肿瘤的生长。. 综上所述，本项目发现了ZBTB7B具有影响小鼠乳腺退化期及肝癌起始的功能，并揭示了其调控机制；同时也发现了COL4A5促进管腔型乳腺癌的功能，为乳腺发育异常、肝癌及乳腺癌研究提供一定的理论基础。