Naive-like重编程状态的诱导多潜能肝癌干细胞研究

Based on previous work found OSKM-mediated reprogramming of C3A human hepatoma cells, induced pluripotent cancer stem-like cells (C3A-iPCs) expressed endogenous reprogramming genes, expressed CD44 and nuclear translocation, maintained “primed” state of iPCs. CD44KO-C3A-iPCs were obtained with Crispr-cas9-mediated CD44 gene knockout in C3A-iPCs, expressed endogenous reprogramming genes in a substantial increase and maintained "naive" –like state of iPCs. Both primed and naive-like states of iPCs we studies its transcriptional regulation and epigenetic memory mechanism. Further, directed hepatic differentiation we will research these cells how to be turned back in naive-like reprogramming state of iPCs. Herein we will study CD44 may be carried the epigenetic memory and does not erase the tumor"self" epigenetic markings in primed state, but in a naive-like reprogramming state CD44 epigenetic information is erased. Expected results both primed and naive states of iPCs may be converted to each other under certain conditions, associated with DNA methylation and histone modification mechanism. The study will propose new ideas and perspectives in clinical tumor therapy.

前期工作发现OSKM介导C3A人肝癌细胞重编程,获诱导多潜能肿瘤干细胞C3A-iPCs,CD44表达并核转位,内源性重编程基因表达,保持肿瘤干细胞Primed状态。Crispr方法敲除CD44基因,获得CD44KO-C3A-iPCs细胞重编程基因的表达大幅度提高,提示保持肿瘤干细胞Naive-like重编程状态。本项目将研究两种不同状态的细胞与转录调节和表观遗传记忆机制。进一步定向肝诱导分化,拟研究Naive-like重编程状态的诱导肿瘤干细胞的转归。证明CD44基因携带表观遗传记忆,Primed状态的诱导多潜能肿瘤干细胞没有擦除肿瘤“自已”的表观标志,而Naive-like重编程状态CD44的表观遗传标志被擦除。本研究将揭示诱导多潜能肿瘤干细胞的Primed和Naive表观遗传状态可在特定的条件下互相转换,与DNA甲基化和组蛋白修饰机制相关。研究将为临床肿瘤治疗提出新的研究思路和前景。

依据文献，疏理前期研究工作，采用转导四个重编程因子Oct4、Sox2、Klf4和c-Myc（OSKM）介导C3A人肝癌细胞（HCC）重编程和Crispr敲除CD44等方法，建立诱导多潜能肝癌干细胞系C3A-iCSCs，发现CD44表达并核转位、内源性重编程基因表达、保持肿瘤干细胞状态。基于此，我们研究了诱导多潜能肝癌干细胞表型的转录调控和表观遗传调节的分子机制。探讨肿瘤发生中肝癌细胞的“干性”和多潜能性与基因表达事件之间的重要分子事件，包括①抑制表观遗传调节相关的染色质重塑复合体基因MBD3表达，提高肝癌干细胞（shMBD3-iCSCs）重编程效率；②shMBD3-iCSCs中转录因子c-JUN上调，活化多潜能基因，促进上皮间质转化。MBD3结合在c-Jun的启动子上，抑制c-Jun转录。c-JUN结合在多潜能基因Klf5和Zfp42的启动子AP-1位点上，直接激活其转录。c-Jun的启动子H3K9乙酰化增加，转录上调；③基于人类基因表达谱芯片分析和验证发现C3A-iCSCs中CD44蛋白可以结合importin β和transportin 1，CD44内化与importin β和transportin 1的转运功能有关；④Pescadillo（PES1）核仁蛋白与肝癌细胞恶性程度密切相关。CD44通过miRNA作用于PES1的3’UTR区，证实了在肝癌干细胞C3A-iCSCs中CD44通过miR-105-5p调控PES1的表达来促进细胞的增殖；⑤CRISPRi介导CD44沉默后，诱导多潜能肝癌干细胞对NK细胞介导的细胞毒性的敏感性明显下降。CD44 3’UTR主要通过竞争性miR-34a调节ULBP2的表达，调节ULBP2的表达增强肝癌干细胞的NK敏感性。本项目研究为肝癌的临床治疗提供新的研究视角和科研思路。