纤毛重构调控Hedgehog信号-自噬效应链对去分化软骨肉瘤恶性进程的影响及机制研究

Chondrosarcoma is the second malignant bone tumor，Dedifferentiated chondrosarcoma is a distinct form of chondrosarcoma where a high-grade sarcoma is found adjacent to a low grade chondrosarcoma. This aggressive tumor is associated with a poor prognosis and its maligment regulation mechanism is still not clear. Primary cilia, an important part of Hedgehog pathway, can regulate cartilage development, but the mechanism of primary cilia on regulating dedifferentiated chondrosarcoma development is not yet clear. Cilia are important sensors and signal transduction units in cells and mediate the connection between cells and microenvironment. Many researches have shown that cilia deficiency had a close link with tumor malignant processes and involved in regulating Hedgehog-autophagy effect chain to determine cells fate. In our previous study, we have found that primary cilia expressed lower in chondrosarcoma with an abnormal activation of Hedgehog signaling compared with normal cartilage. Induction of cilia remodeling could be associated with an increase of autophagosome and influence chondrsarcoma cells malignant biological behaviors. This project aim to explore the effects on Hedgehog pathway to regulate dedifferentiated chondrosarcoma malignant process by regulating cilia and autophagy expression and identify the potential mechanism of Hedgehog pathway involved in cilia and autophagy interaction. To clarify the role of primary cilia affecting Hedgehog-autophagy effect chain in regulating dedifferentiated chondrosarcoma development and malignant process will provide new theoretical basis and experimental basis to find a new treatment of chondrosarcoma based on the different expression of primary cilia.

软骨肉瘤是第二常见的恶性骨肿瘤，其中去分化软骨肉瘤恶性程度高、发展快、缺乏有效的治疗手段，其恶性生物学行为的调控机制尚不明确。初始纤毛作为Hedgehog通路重要的信号传导单元参与调控软骨发育，同时初始纤毛还是重要的细胞感受器介导细胞和微环境之间的联系，有证据显示纤毛缺失与肿瘤的恶性进程密切相关，且参与调控Hedgehog-自噬效应链进而左右细胞命运。申请者在前期研究中发现软骨肉瘤中存在纤毛的低表达，同时伴有Hedgehog通路的异常激活，诱导纤毛重构可伴有自噬上调并影响软骨肉瘤的恶性生物学行为。本项目拟通过探究可调控纤毛与自噬表达对去分化软骨肉瘤Hedgehog通路及肿瘤恶性进程的影响，明确Hedgehog通路参与调控纤毛与自噬交互作用的潜在机制，阐明纤毛影响Hedgehog-自噬效应链在去分化软骨肉瘤恶性进程中的作用，为寻找基于纤毛表达差异的软骨肉瘤治疗新方法提供理论和实验基础。

本项目主要研究软骨肉瘤中Hedgehog相关信号及纤毛的交互作用对软骨肉瘤的影响，首先检测Hedgehog(HH)信号通路在人软骨肉瘤中的表达情况，探讨HH信号通路抑制剂HPI-4对于软骨肉瘤细胞SWl353增殖和侵袭能力的影响。经不同浓度HPI-4干干预后，证实肿瘤细胞侵袭能力随着HPI-4浓度的增高而递减；Western blot结果显示肿瘤细胞内增殖与侵袭相关蛋白的表达明显下降。然后探讨Hedgehog信号下游PTH通路对软骨肉瘤细胞增殖和侵袭能力的影响。结果 PTH 能促进软骨肉瘤细胞增殖,且呈浓度依赖性,其促增殖效应与纤毛结构的完整性有关;,破坏纤毛结构后,PTH 促进软骨肉瘤的侵袭效应受到抑制；PTH 能调控 Hedgehog通路靶基因 GLI1、PTCH1及纤毛功能相关基因IFT88的表达。为了检测人软骨肉瘤原发性纤毛与HDAC6的相互作用，使用Tubastatin A和小干扰RNA (small RNA, siRNA)抑制HDAC6的内源性表达。评价恶性生物学特性的影响。检测原发性纤毛染色及相关蛋白。发现软骨肉瘤组织中HDAC6和纤毛内转运蛋白 IFT88表达异常。HDAC6的抑制可以浓度依赖性和时间依赖性的下调软骨肉瘤细胞的增殖，抑制肿瘤细胞的侵袭能力。综上所述，本研究发现人软骨肉瘤中存在HH信号通路的明显激活，HH信号通路抑制剂HPI-4能够明显抑制软骨肉瘤细胞的增殖和侵袭能力。Hedgehog信号通路下游 PTH信号 能促进软骨肉瘤细胞的增殖和侵袭,同时抑制软骨肉瘤细胞初级纤毛的表达并影响其，下游靶基因表达,PTH 可能通过作用于初始纤毛来调控软骨肉瘤的恶性生物学特性。限制HDAC6可以恢复原发性纤毛组装，抑制软骨肉瘤细胞的增殖和侵袭能力。因此，靶向HDAC6促进原发性纤毛修复可能是一种可行的潜在软骨肉瘤治疗方法。