发育多能性相关蛋白DPPA3通过调控GLI1启动子区DNA甲基化促进肝癌干细胞自我更新的功能和机制研究

Self-renewal of cancer stem cells is the major cause of tumor development, invasion and metastasis. Through in vitro hepatocyte differentiation model analysis, our previous study showed developmental pluripotency associated protein 3 – DPPA3 was highly expressed in liver progenitor stage, indicating its role in both liver tissue and liver cancer stem cells. Our preliminary data demonstrated that DPPA3 expression was evidently higher in poor-differentiated HCC tissues, compared with well differentiated ones. Besides, upregulation of DPPA3 could enhance cells capabilities of sphere formation and anti-apoptosis. Integrated analysis of RNA-seq and Bisulfite Genomic Sequencing indicated transcription factor GLI1 is the downstream target of DPPA3, which could promote HCC cells sphere formation capability. Furthermore, DPPA3 was able to bind with epigenetic regulator UHRF1, which may lead to DNA methylation alteration. Thus, we propose a hypothesis that DPPA3 promotes the self-renewal of cancer stem cells by interacting with UHRF1 and regulating DNA methylation in the promoter region of GLI1. To test our hypothesis, we prepare to conduct the following studies. Firstly, the association between DPPA3 expression and tumor differentiation in the clinical samples is to be analyzed. Secondly, whether DPPA3 could promote the self-renewal of liver cancer stem cells is to be verified both in vitro and in vivo. Then, experiments such as CoIP and bisulfite genomic sequencing (BGS) will be applied to investigate the underlying mechanism of how DPPA3 alters DNA methylation pattern in the promoter region of GLI1. Through this study, we may provide a new method to treat and prevent HCC recurrence via targeting the expression of DPPA3.

干细胞的自我更新与肝癌发生发展密切相关。由于肝癌干细胞与肝脏干细胞具有相似调控网络，我们通过前期构建体外人源肝细胞分化模型，筛选到在肝祖细胞阶段高表达的发育多能性相关蛋白DPPA3。其表达水平与肝癌分化程度成反比；上调其表达可增强细胞悬浮成球和抗凋亡能力；多组学测序发现DPPA3的下游靶基因为转录因子GLI1；GLI1可增强细胞悬浮成球能力；还发现DPPA3可与表观遗传调控因子UHRF1结合，影响DNA甲基化水平。据此我们提出假设，DPPA3与UHRF1互作调控GLI1启动子区DNA甲基化，从而促进肿瘤干细胞自我更新。为验证该假设，本项目拟扩大肝癌临床标本，证实DPPA3与肿瘤分化的相关性；然后，利用体内外实验明确DPPA3可促进肝癌干细胞自我更新；最后，通过CoIP、BGS等方法，重点探讨DPPA3调控GLI1启动子区DNA甲基化的机制。本项目有望为靶向肿瘤干细胞治疗策略提供实验依据。

肝癌干细胞是一群数量稀少的具有自我更新能力的癌细胞群。然而，调控细胞层次和干性的分子事件在很大程度上仍然未知。在本项研究中，基于对体外肝细胞分化模型的分析，我们发现DPPA3在肝祖细胞和肝癌干细胞中均呈高表达。功能研究表明DPPA3可增进肝癌细胞系或肝癌类器官的干性特征，并促进其发生化疗、靶向耐药。由于DPPA3可通过影响DNA甲基化水平来调控下游基因表达，我们在DPPA3转染的肝永生化细胞系MIHA中进行了全基因组甲基化测序与RNA测序的联合分析，鉴定出DPPA3的下游靶标为GLI1和MYCN。接下来的分子机制研究发现，GLI1在DPPA3介导的干性调控中具有关键作用。采用shRNA敲低GLI1或利用小分子抑制剂干扰GLI1活性，可显著下调MYCN表达，并消除DPPA3对肝癌干细胞的干性调控作用。此外，我们发现DPPA3可与表观调控因子UHRF1结合并抑制其核定位，导致GLI1和MYCN启动子区发生DNA去甲基化。最后，小鼠体内实验发现GLI1抑制剂与索拉非尼联合治疗可协同抑制小鼠皮下肿瘤生长。因此，本项目发现DPPA3通过调控DNA甲基化水平介导GLI1/MYCN激活，促进并维持肝癌干细胞的干性特征，最终导致治疗耐药、转移复发和不良预后。靶向并阻断DPPA3/GLI1/MYCN通路可能是一种有希望逆转肝癌治疗耐药和防治肿瘤复发的治疗策略。

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