BACKGROUND Liver cancer stem cells (CSCs) are critical determinants of HCC relapse and therapeutic resistance, but the mechanisms underlying the maintenance of CSCs are poorly understood. We aimed to explore the role of tumor repressor Zinc-fingers and homeoboxes 2 (ZHX2) in liver CSCs. METHODS CD133+ or EPCAM+ stem-like liver cancer cells were sorted from tumor tissues of HCC patients and HCC cell lines by flow cytometry. In addition, sorafenib-resistant cells, tumor-sphere forming cells and side population (SP) cells were respectively cultured and isolated as hepatic CSCs. The tumor-initiating and chemoresistance properties of ZHX2-overexpressing and ZHX2-knockdown cells were analyzed in vivo and in vitro. Microarray, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and ChIP-on-chip analyses were performed to explore ZHX2 target genes. The expression of ZHX2 and its target gene were determined by quantitative RT-PCR, western blot, immunofluorescence and immunohistochemical staining in hepatoma cells and tumor and adjacent tissues from HCC patients. RESULTS ZHX2 expression was significantly reduced in liver CSCs from different origins. ZHX2 deficiency led to enhanced liver tumor progression and expansion of CSC populations in vitro and in vivo. Re-expression of ZHX2 restricted capabilities of hepatic CSCs in supporting tumor initiation, self-renewal and sorafenib-resistance. Mechanically, ZHX2 suppressed liver CSCs via inhibiting KDM2A-mediated demethylation of histone H3 lysine 36 (H3K36) at the promoter regions of stemness-associated transcription factors, such as NANOG, SOX4 and OCT4. Moreover, patients with lower expression of ZHX2 and higher expression of KDM2A in tumor tissues showed significantly poorer survival. CONCLUSION ZHX2 counteracts stem cell traits through transcriptionally repressing KDM2A in HCC. Our data will aid in a better understanding of molecular mechanisms underlying HCC relapse and drug resistance.

中文翻译：

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ZHX2通过通过KDM2A介导的H3K36去甲基化抑制干细胞样性状来限制肝癌。

背景技术肝癌干细胞（CSC）是HCC复发和治疗抗性的关键决定因素，但对CSC维持的基本机制了解甚少。我们旨在探讨肿瘤抑制因子锌指和同源盒2（ZHX2）在肝CSC中的作用。方法采用流式细胞术从肝癌患者的肿瘤组织和肝癌细胞株中分离出CD133 +或EPCAM +干细胞样肝癌细胞。另外，分别培养和分离索拉非尼抗性细胞，形成肿瘤球的细胞和侧群（SP）细胞作为肝CSC。体内和体外分析了ZHX2过表达和ZHX2敲除细胞的肿瘤启动和化学耐药性。芯片，荧光素酶报告基因检测，进行了染色质免疫沉淀（ChIP）和芯片上芯片分析，以探索ZHX2靶基因。通过定量RT-PCR，western blot，免疫荧光和免疫组化染色确定ZHX2及其靶基因在肝癌患者肝癌细胞和肿瘤及癌旁组织中的表达。结果不同来源的肝CSC中ZHX2表达明显降低。ZHX2缺乏导致体外和体内CSC人群肝脏肿瘤进展增强和扩展。ZHX2的重新表达限制了肝CSC在支持肿瘤起始，自我更新和索拉非尼耐药性方面的能力。在机械上，ZHX2通过抑制KDM2A介导的与干性相关的转录因子（如NANOG）的组蛋白H3赖氨酸36（H3K36）的去甲基化来抑制肝脏CSC，SOX4和OCT4。此外，在肿瘤组织中ZHX2表达较低而KDM2A表达较高的患者，其生存期明显较差。结论ZHX2通过在肝癌中转录抑制KDM2A来抵消干细胞性状。我们的数据将有助于更好地了解HCC复发和耐药性的分子机制。