CBX4在Bmp2/Smad/Runx2介导的颅咽管瘤钙化中的作用和机制研究

Craniopharyngioma is one of the most common tumors in the brain. It is difficult to treat and has a high mortality rate. More than 70% of craniopharyngioma with calcification. Calcification is the key reason for increasing the difficulty of surgery for craniopharyngioma. However, the underlying mechanism still remains to be elucidated. Studies have found that Bmp2/Smad induced Runx2 transcription activation is a key mechanism for promoting ossification and calcium deposition, and histone deacetylase 3 (HDAC3) can inhibit Runx2 expression. Our research found that Bmp2 induces high expression of Runx2 in craniopharyngioma cells and also promotes HDAC3 expression. Further preliminary experiments revealed that polycomb protein 4 (CBX4), which stably binds HDAC3 to the Runx2 promoter, has reduced expression in Bmp2-induced craniopharyngioma cells.Therefore, it is speculated that Bmp2 may reduce the inhibition of Runx2 by HDAC3 by inducing the decrease of CBX4 expression, and promote calcification. This project is intended to combine the in vitro and in vivo experiments, using protein overexpression, gene knockout and other techniques to clarify the regulatory mechanism of CBX4 stable HDAC3 on the activity of Bmp2/Smad/Runx2 axis, explore the mechanism of Bmp2-induced low expression of CBX4 and elucidate the mechanism of Bmp2-induced calcification of craniopharyngioma. And provide the necessary theoretical basis for exploring new surgical adjuvant therapy for this disease.

颅咽管瘤是最常见的颅内肿瘤之一，其治疗难度大，死亡率高；超过70%的颅咽管瘤合并钙化，钙化是造成其手术难度大的关键因素，而钙化的机制尚不明确。有研究认为骨形成蛋白2(Bmp2)/Smad介导的Runx2转录激活可以促进成骨及钙盐沉积；而组蛋白去乙酰化酶3(HDAC3)可抑制Runx2的转录。我们的研究发现，Bmp2可诱导颅咽管瘤细胞的钙盐沉积，但观察到HDAC3和Runx2的表达都升高；而稳定HDAC3结合到Runx2启动子的CBX4表达降低；因此推测Bmp2可能通过诱导CBX4表达降低，解除了HDAC3对Runx2的抑制，促进了钙化。本项目拟结合体内外实验，利用蛋白过表达、基因敲除等技术，明确CBX4稳定HDAC3对Bmp2/Smad/Runx2轴活性的调控机制；探讨Bmp2诱导CBX4低表达的机制；最终阐明Bmp2诱导的颅咽管瘤钙化的机理，为探索本病新的手术辅助疗法提供必要的理论依据。

严重钙化的颅咽管瘤(ACP)导致切除肿瘤的风险相当高。组蛋白乙酰转移酶(hAT)和组蛋白脱乙酰酶(HDAC)是调节组蛋白乙酰化的两种关键酶，在肿瘤发展中起着重要作用。然而，HAT 和 HDAC 在 ACP 钙化中的作用尚不清楚。在本研究中，我们发现在钙化的 ACP 组织中 HDAC3的表达增加，但是抑制 HDAC3的表达促进 ACP 钙化。Bmp2抑制 HDAC3核移位，增加 Runx2蛋白表达，以及 Osterix，OCN，OPN 和 ALP mRNA 和 miR-181b 水平。小型泛素样修饰酶(SUMO) E3连接酶 CBX4稳定了 HDAC3的核定位，并降低了 Runx2蛋白表达和 Osterix、 OCN、 OPN 和 ALP mRNA 水平，以及 ACP 细胞钙化。MiR-181b 直接靶向并降低 CBX4的表达。总之，Bmp2增加 miR-181b 水平以直接靶向和抑制 CBX4表达，导致 CBX4依赖性调节 HDAC3核易位减少和抑制 HDAC3 Runx2信号传导途径。针对这种级联反应的进一步研究可能有助于未来复发性 ACP 的治疗干预。