Msi2基因调控与Wnt/β-catenin和Notch信号通路间串话在CD44＋肝癌干细胞“干性”维持中的作用

The stem cell marker CD44 had been demonstrated in previous our studies to be closely related to invasion, metastasis and relapse of hepatocellular carcinoma(HCC). Msi2 is a potential regulatory gene that is involved in the maintenance of cancer stem cell's "stemness". Wnt and Notch signaling pathway have been reported to be essential for maintaining the stemness of liver cancer stem cells such as the capacity of self-renewal, invasion and tumorigenicity. Our project aims to detect the different expression of Msi2 gene in purified CD44 ＋/－ cells which have been isolated from cell lines and clinical HCC tissue and have been identified by functional in vitro clonogenicity and in vivo tumorigenicity assays. Moreover, the influence of Msi2 gene on the stem-cell-related characteristics will be tested in CD44 + cells by RNAi method and in CD44-cells by ectopic expression of Msi2. The main purpose of the present study is to clarify the key role of Msi2 gene in maintaining CD44＋ liver cancer stem cell's "stemness" features. Furthermore, we will detect the changes in CD44－ cells' stem-cell-related characteristics and the expression of Msi2 after up-regulating the Wnt signaling and/or down-regulating the Notch signaling. Finally, we will employ CHIP assay to test the potential cross-talk bewteen Wnt and Notch signaling which may be regulated by the Msi2 and maintain the stemness of CD44 + liver cancer stem cells. From the aspect of cross-talk between signaling networks, the regulation of stemness-related gene and maintenance of liver cancer stem cell's "stemness" , we will provide a new clue for the mechanisms of invasion and metastasis and recurrence of HCC, which is expected to warrant a new target for treatment of HCC.

前期研究显示新近发现的"干性"标记CD44与肝癌的侵袭、复发密切相关，Msi2是一种潜在的维持肿瘤干细胞"干性"的调控基因，Wnt与Notch信号通路在维持肝癌干细胞的自我更新、侵袭与致瘤能力等"干性"特征方面至关重要。本项目拟先检测经分选与鉴定的CD44＋/－细胞间Msi2基因表达的差异，再观察通过慢病毒载体介导的Msi2上调与下调对CD44＋/－细胞"干性"特征的影响，试图阐明Msi2在维持CD44＋细胞"干性"中的关键作用；进一步观察调控Wnt和Notch信号通路后CD44＋细胞"干性"及Msi2表达的变化，并采用序贯调控及CHIP实验研究Wnt与Notch信号通路间可能的串话作用机制，探讨CD44＋细胞Msi2基因表达的调控与"干性"维持的分子机制，为从信号网络间串话及干性基因调控与肝癌干细胞"干性"维持这一角度研究肝癌侵袭与复发机制提供新的思路，可望提供肝癌生物治疗的新靶点。

肝癌干细胞与肝癌的复发、转移密切相关，且有多个表面标记物，如EpCAM、CD90、CD24、CD133等，但CD44v6是否可作为肝癌干细胞表面标记物的报道较少。Msi2基因在肿瘤干细胞的“干性”维持中发挥着重要作用，研究显示Notch信号通路可参与调节肿瘤干细胞的“干性”特征，但Msi2基因与Notch信号通路在肝癌干细胞中的作用鲜有报道。本项目通过检测肝癌组织标本中CD44v6、Msi2的表达，发现Msi2在肝癌组织中表达明显高于癌旁组织，且Msi2高表达与肿瘤分化密切相关。采用免疫磁珠分选法成功从肝癌细胞系MHCC-97H、SNU398中分选出CD44v6+/CD44v6-细胞，通过一系列的“干性”鉴定实验证实CD44v6+细胞是一群具有自我更新、致瘤、转移能力强、化疗抵抗等“干性”特征的细胞亚群。在检测CD44v6+细胞的Msi2表达明显高于CD44v6-细胞的基础上，进一步通过慢病毒下调/上调CD44v6+/-细胞的Msi2后观察细胞生物学行为变化和Notch信号通路关键分子表达的变化,发现Msi2在CD44v6+肝癌干细胞“干性”维持中发挥重要作用，并与Notch通路正相关。采用蛋白阻遏和慢病毒调控Notch信号通路进一步证实了Notch信号通路在CD44v6+肝癌干细胞“干性”维持中的关键作用。最后通过信号通路基因芯片和免疫共沉淀的机制研究发现Msi2基因可能通过Lfng分子激活Notch信号通路参与CD44v6+肝癌干细胞的“干性”维持作用。总之，本课题确定了CD44v6为肝癌干细胞重要的“干性”表面标记，并阐述了Msi2通过Lfng活化Notch信号通路参与在CD44v6+肝癌干细胞“干性”维持的作用及其可能的分子机制，为研究肝癌侵袭与复发机制提供了新的思路，可望为肝癌的分子靶向治疗提供潜在的分子靶点，进一步优化肝癌的分子靶向治疗策略。