Hippo-YAP信号通路对正常造血干/祖细胞生物学特性和功能调控以及和白血病发病机制关系的实验研究

Hippo-YAP is a newly discovered signal transduction pathway, of which YAP is the core protein. YAP is a transcription co-activator that regulates cluster of genes associated with cell proliferation,differentiation and apoptosis. Thus, it controls homoeostasis of stem cell and organ regeneration. A dysfunction of the pathway is frequently detected in human cancers. However, there was no study that directly addressed whether or not and how the canonical Hippo pathway functions in hematopoietic stem cells(HSC)/ progenitor cells(HPC) and leukemia cells. In our previous study, we have detected certain expression of genes belong to Hippo-YAP pathway in normal HSC and leukemia cells, but their functions are still unclarified. Thus, the goals of this project are to:(1) accurately profile expression levels of main components of Hippo-YAP pathway in the HSC/HPC;(2)ectopic gain-of-function overexpression or shRNA intervened loss-of-function knockdown of YAP in murine HSC/HPC, examine HSC/HPC capacity of proliferation, apoptosis and differentiation, respectively. in addition, unveil YAP regulated possible downstream target genes related to maintain HSC/HPC self-renewal ability via CHIP teqnique;(3)construction YAP and BCR/ABL co-expressed murine HSC transplantation model, investigate influence of YAP on leukemogenesis. This project will pave new way for better understanding of hematopoiesis regulation and leukemia-targeted therapy.

Hippo-YAP是新信号通路，YAP为效应因子，属转录共激活物调控多种与细胞增殖、分化、凋亡相关基因,并与肿瘤发生相关。我们前期研究发现该通路分子在正常造血细胞和白血病细胞中均有表达，但功能及意义不详。故本课题拟从分子，细胞及动物实验层面深入开展研究：(1)定量检测Hippo-YAP通路中主要基因在小鼠造血干/祖细胞（HSC/HPC）中的表达水平；(2)实现YAP在鼠HSC/HPC中功能获得性过表达（gain-of-function overexpression）及基因沉默（loss-of-function knockdown），分别观察HSC/HPC生物学特性及功能变化。CHIP实验进一步寻找受YAP调控的下游靶基因；(3)建立YAP基因和BCR/ABL基因共表达小鼠干细胞移植模型，研究Hippo通路和慢性粒细胞白血病发病机制的关系。本课题为深入了解造血调控及白血病靶向治疗提供新思路。

一.项目背景：Hippo-YAP 是新信号通路，YAP 为效应因子，属转录共激活物调控多种与细胞增殖、分化、凋亡相关基因,并与肿瘤发生相关。Hippo-YAP通路在正常造血调控以及造血系统疾病中的功能及意义目前尚无相关研究。因此，本课题对上述科学问题展开深入研究。.二.主要研究内容：(1)定量检测Hippo-YAP 通路中主要基因在小鼠造血干/祖细胞（HSC/HPC）中的表达水平；(2)实现YAP 在鼠HSC/HPC 中过表达及基因沉默，分别观察HSC/HPC 生物学特性及功能变化。CHIP 实验进一步寻找受YAP 调控的下游靶基因；(3)建立YAP 基因和BCR/ABL基因共表达小鼠干细胞移植模型，研究Hippo 通路和慢性粒细胞白血病发病机制的关系。.三.重要结果、关键数据及其科学意义：.1.realtime-PCR精确定量Hippo-YAP通路中主要基因Merlin、 Sav1、MST1/2、 LATS1/2和YAP在小鼠正常造血干/祖细胞（HSC/HPC）中的表达水平。结果发现Hippo-YAP通路中主要基因Merlin、 Sav1、MST1/2、 LATS1/2和YAP在小鼠正常造血干/祖细胞（HSC/HPC）中均有不同程度的表达，提示该信号通路在造血干/祖细胞功能调控中可能发挥了一定作用。.2.成功构建了Hippo-YAP通路中最关键基因—YAP基因在鼠HSC/HPC中功能获得性过表达（gain-of-function over-expression）的逆转录病毒载体及慢病毒表达载体，.并转染了小鼠正常造血干/祖细胞（HSC/HPC），发现YAP基因过表达能够增强造血干/祖细胞集落形成能力。提示该信号通路参与造血干/祖细胞的增殖过程。.3.探索了Hippo-YAP通路中最关键基因—YAP基因在常见血液系统恶性肿瘤细胞系中的表达情况。结果发现人T淋巴细胞白血病细胞系Jurkat中存在YAP基因异常高表达，利用RNA干扰实验技术，通过慢病毒感染Jurkat细胞，实现了在Jurkat细胞系中沉默YAP基因，结果发现干扰慢病毒pL/ShRNA-yap-1522可明显引起Jurkat靶细胞凋亡。并在一定程度上将Jurkat细胞阻滞在G1/S期。提示Hippo-YAP信号传导通路在人T淋巴细胞白血病细胞的增殖及凋亡中发挥重要作用。