Hedgehog-Gli1信号通路调控Nanog参与宫颈癌干细胞自我更新中的作用及机制研究

Self-renewal is the important biological characteristics of cancer stem cells (CSCs), and inhibition of self-renewal regulator is an effective way to control tumor growth. On the past study shown that Hedgehog-Gli1 were highly expressed in uterine cervical cancer tissue and cell lines, and to further promoted the invasion and metastasis of cervical cancer cells by epithelial mesenchymal transition. There was higher expression of Gli1 and Nanog were observed in cervical cancer cells with hypoxia condition, and the effects of hypoxia cancer cells in sphere formation and tumorigenicity in nude mice were significantly higher than normoxia cells. However, the specific biological function of Hedgehog-Gli1 regulate cervical cancer stem cells and the molecular mechanisms of transcriptional regulation were remains unclear. The project intends to use bioinformatical analysis and molecular biological technic to clarify the effects of Hedgehog-Gli1 in cervical cancer stem cells self-renewal at cells, animal and clinical specimens level in all-round, muti-levels. And than to explore Hedgehog-Gli1 molecular mechanisms in CSCs self-renewal formation through activate Nanog expression in transicription levels, and to provide new molecular targets and strategies for cervical CSCs treatments.

自我更新是肿瘤干细胞( cancer stem cells，CSCs)的重要生物学特性之一，抑制自我更新调节器是控制肿瘤生长的有效途径。前期研究表明，Hedgehog-Gli1通路基因在宫颈癌组织和细胞高表达，通过上皮间质转化进一步促进宫颈癌细胞的侵袭转移能力。缺氧诱导的宫颈癌细胞中Gli1和Nanog表达均明显增高；缺氧宫颈癌细胞的球形体形成能力和裸鼠皮下成瘤能力明显高于常氧细胞。但Hedgehog-Gli通路调控宫颈癌CSCs的具体生物学功能及其转录调控分子机制仍不清楚。本项目拟采用生物信息分析和分子生物学技术，在细胞、动物和临床标本水平上全方位、多层次阐明Hedgehog-Gli通路在宫颈癌CSCs自我更新中的作用，探讨Hedgehog-Gli1通路通过转录水平激活Nanog的表达参与CSCs自我更新形成的分子机制，为宫颈癌CSCs的治疗提供新的分子靶点和策略。

肿瘤干细胞与宫颈癌的起源和进展密切相关，肿瘤干细胞成为宫颈癌治疗和干预的重点。本项目采用免疫组织化学染色法观察了161例宫颈癌组织中Hedgehog （Hh）信号通路关键因子（SMO和Gli1）的表达，研究结果表明宫颈癌组织中SMO与Gli1的表达均与病理学分期、远期转移以及HIF1α的表达密切相关；Gli1阳性组微血管密度明显高于阴性组。在宫颈癌组织中SMO的表达与p21，pAkt-Thr308蛋白表达呈正相关关系；而Gli1的表达与p21表达呈正相关关系。宫颈癌组织中Gli1的表达与Sox2蛋白的表达呈正相关关系。宫颈癌缺氧细胞模型中，SMO和Gli1的mRNA及蛋白的表达高于常氧组细胞，缺氧诱导宫颈癌细胞发生上皮间质转化，并且缺氧组的肿瘤干细胞标志因子Oct-4、Sox2和Nanog 的mRNA表达水平明显提高。体外球形体形成实验结果示，缺氧组球形体形成能力明显高于常氧组。球形体消化后接种于裸鼠皮下，球形体细胞在体内成瘤能力明显强于非球形体细胞。Gli1抑制剂GANT61使宫颈癌HeLa细胞E-Cadherin蛋白表达水平提高，而Snail及Vimentin蛋白表达水平降低；肿瘤干细胞标志蛋白oct-4、sox2和Nanog的表达水平均降低。Gli1抑制剂GANT61明显降低HeLa细胞的侵袭迁移能力及裸鼠体内成瘤能力。本项目的研究结果揭示了Hedgehog-Gli1在宫颈癌进展及预后的临床病理学意义，阐明了该通路与自我更新等肿瘤干细胞特性的相关性，可能为宫颈癌的治疗提供新的靶点。