异常活化的Hh信号通路促进卵巢癌侵袭转移的分子机制研究

Ovarian cancer is a highly aggressive cancer, and there is no specific treatment for it. Recent studies suggest that aberrant activation of Hh signaling is implicated in ovarian cancer. However, the molecular mechanisms of extremely activated Hh signaling pathway promoting cell invasion and metastasis in ovarian cancer are not well understood.Our previous study showed that inhibition of Hh signaling pathway may reduce ovarian cancer migration and invasion. Gene expression profiling of Hh signaling inhibited ovarian cancer cells was determinated by cDNA microarray. Results indicated that genes promotng cell adhesion, migration and invasion in ovarian cancer cells were significantly differentially expressed. All of the above-mentioned suggested that genes such as ITGB4,MMP7,FAK etc regulated by Hh signaling pathway play an important role in tumor invasion and matestasis of ovarian cancer. Based on these previous works, this project will discover new genes regulated by Hh signaling pathway. Multiple levels from the cellular level, the molecular level, animal models and clinical specimens, to clarify the role of the new target genes regulated by the Hh signaling pathway, further reveal The molecular mechanisms research of extremely activated Hh signaling pathway promoting cell invasion and metastasis in ovarian cancer. Our final objective is to Provide a theoretical basis for ovarian cancer targeted therapy.

卵巢癌是高侵袭性恶性肿瘤，缺乏特异性诊疗手段。近年研究发现，卵巢癌中Hedgehog(Hh)信号通路异常活化，但相关机制以及异常活化后促进卵巢癌侵袭转移的分子机制有待明晰。我们的前期研究发现，抑制Hh信号通路可降低卵巢癌细胞迁移侵袭能力；利用基因表达谱芯片等技术我们进一步发现，与卵巢癌细胞粘附、迁移及侵袭相关的基因如ITGB4, MMP7, FAK等在Hh信号通路抑制剂处理的细胞中发生显著改变(Q.Chen, et al. PLoS ONE 2014; 9(2): e88386),提示Hh信号通路调控的基因在卵巢癌侵袭及转移过程中可能发挥重要作用。本项目拟进一步发掘受Hh信号通路调控的靶基因；从细胞水平、分子水平、动物模型及临床队列等几个层次，发掘Hh信号通路调控的新基因及其功能，进一步揭示异常活化的Hh信号通路促进卵巢癌侵袭转移的分子机制，为卵巢癌靶向治疗提供理论依据和新思路。

卵巢癌是高侵袭性恶性肿瘤，缺乏特异性诊断方法。近年研究发现，卵巢癌中Hedgehog(Hh)信号通路异常活化，已有研究发现抑制Hh信号通路可降低卵巢癌细胞迁移侵袭能力,但是相关的分子机制尚未明了。本次研究中，我们主要从细胞、分子、动物水平及临床队列等几个层次探索其分子机制。N-Shh激活卵巢癌细胞Hh信号通路通过Western-blot 及RT-PCR实验发现CD24、MMP-7表达上调，且随时间梯度上调，Hh信号通路抑制剂Cyclopamine和（或）GANT61作用卵巢癌细胞系后通过Western-blot 及RT-PCR实验发现CD24、MMP-7表达下调，且随时间梯度下调，由此猜测CD24及MMP-7是Hh信号通路下游靶基因。通过南昌大学第二附属医院临床样本研究发现，CD24在卵巢癌组织中异常高表达。构建了CD24干扰质粒，通过划痕实验及Transwell实验发现，下调CD24后抑制了N-Shh对卵巢癌细胞迁移和侵袭的刺激作用。裸鼠成瘤实验发现GANT61能明显抑制裸鼠皮下成瘤能力，剥出裸鼠肿瘤，免疫组化实验发现，GANT61组肿瘤组织CD24表达明显下降。因此得出结论，Hedgehog信号通路通过粘附分子CD24调控卵巢癌的侵袭和迁移。通过染色质免疫共沉淀技术（CHIP实验）检测Hedgehog信号通路转录因子Gli与MMP-7基因启动子区的结合情况，发现在特异性Gli2抗体免疫沉淀的DNA片段中扩增出MMP-7基因启动子序列，说明转录因子Gli2可以与MMP-7基因的启动子的特异区域结合，验证了MMP-7是Hh下游直接靶基因。成功构建MMP-7干扰质粒，通过划痕实验及Transwell实验发现干扰MMP-7能促进卵巢癌细胞迁移和侵袭，可以减弱N-Shh对卵巢癌迁移和侵袭的刺激作用。通过免疫组化实验发现，Gli2及MMP-7在卵巢癌组织中高表达，且呈正相关。结合公共数据库分析，Gli2及MMP-7高表达与卵巢癌临床分期、病理分级及预后密切相关。Hh-Gli-MMP7信号轴的异常活化对促进卵巢癌恶化和侵袭起重要作用，提示其可能成为卵巢癌的一种新的治疗靶点。MMP-7有望成为卵巢癌的预后标志物。