Rfx6在肝癌干细胞干性特征维持中的作用及机制研究

Embryonic development associated-genes have recently been gained wide attention due to involving in the regulation of cancer stem cells (CSC). Regulatory factor x6 (Rfx6) was identified as a new member of Rfx family, and regulated the differentiation of pancreatic islet cell. Our previous study showed that Rfx6 mRNA expression was positively correlated with hepatic precursor cell markers, and higher Rfx6 mRNA expression was associated with poor differentiation of tumors and worse prognosis in hepatocellular carcinoma (HCC) patients after partial hepatectomy. In addition, overexpression Rfx6 led to active ERK, increase expression of β-catenin, and promote proliferation, migration, invasion and chemoresistance in HCC cell lines. Therefore, we hypothesized that Rfx6 may involve in the maintaining HCC stemness and the Rfx6-ERK-β-catenin pathway may play a role in regulating the self-renew in HCC cell. To verify it, we will first use promoter reporter system, animal model, and sphere formation assay and other experiments to illustrate the correlation of Rfx6 with HCC stemness in Rfx6pos/Rfx6neg HCC cells. Viral-mediated overexpression/reduction of Rfx6 expression cell lines will be constructed to explore the role of Rfx6 in regulation of HCC stemness. Furthermore, ERK inhibitor will also be used to investigate the role of Rfx6-ERK-β-catenin pathway in maintaining stemness of HCC, and ChIP-seq will be employed to identify genes potentially regulated by Rfx6. Our study will help to unveil the role and mechanisms of Rfx6 involved in HCC stemness. In addition, this study will provide new insight for target therapy of HCC.

胚胎发育相关基因可参与肿瘤干细胞的调控。前期研究发现胰岛发育相关基因Rfx6与肝前体细胞标记物的表达相关；肝癌组织中Rfx6高表达提示肝癌分化差及不良预后；Rfx6过表达可激活ERK蛋白并上调β-catenin表达，促进肝癌细胞增殖、迁移、侵袭及化疗抵抗。因此，我们推测Rfx6可能与肝癌干细胞特征相关。本研究拟：⑴通过Rfx6启动子报告系统、小鼠肝原位移植瘤模型、干细胞球形成等实验，探讨Rfx6与肝癌干细胞干性特征的关系；⑵构建Rfx6表达上调/下调的肝癌细胞模型，进一步探讨Rfx6对肝癌干细胞干性特征的调控作用；⑶结合ERK抑制剂，了解Rfx6-ERK-β-catenin通路对维持肝癌干细胞干性特征的作用，通过ChIP-seq等筛选Rfx6调控的下游基因，探讨Rfx6调控肝癌干细胞干性特征的机制。本课题旨在更深入了解肝癌干细胞的特征，为寻找潜在分子靶点提供新的依据。

背景.肝细胞癌是一种具有高侵袭和转移能力、术后易复发、机制研究尚不清楚的肿瘤。本课题组前期研究发现Rfx6与肝前体细胞标记物的表达相关并促进肝细胞癌细胞的迁移、侵袭。本研究旨在进一步阐明Rfx6在肝细胞癌中的功能及机制；明确Rfx6在肝细胞癌预后预测中的作用。.主要研究内容.qRT-PCR、IHC和Western blotting用于检测Rfx6在肝细胞癌组织及细胞中的表达情况；干细胞球形成、qRT-PCR等实验探讨Rfx6与肝癌干细胞干性特征的关系；采用Transwell实验明确Rfx6对肝细胞癌细胞迁移及侵袭能力的影响；通过免疫荧光和 western blotting 观察其对细胞形态和EMT的影响；利用ChIPseq和RNAseq筛选出Rfx6调控的靶基因；利用ChIP、荧光素酶报告等实验研究Rfx6与靶基因之间的相互作用；表达Rfx6后加入靶基因的单克隆抗体，通过Transwell实验分析其对肝细胞癌细胞迁移和侵袭的影响；构建肝原位种植瘤转移模型和肺转移瘤模型研究其在体内对肝细胞癌细胞转移的影响；通过Kaplan-Meier生存分析、单/多因素COX回归分析研究Rfx6与肝细胞癌临床预后之间的关系。.重要结果和关键数据.qRT-PCR、IHC显示Rfx6在肝细胞癌中的表达显著增强；干细胞球形成等实验结果显示Rfx6与与肝癌干细胞干性特征呈正相关；Transwell实验表明Rfx6显著促进细胞的迁移和侵袭；过表达Rfx6后，肝细胞癌上皮细胞呈现EMT样改变；ChIPseq和RNAseq、免疫荧光等实验均证实Rfx6可促进IL6介导的细胞的EMT；ChIP研究表明Rfx6可与IL6启动子区相互作用；ChIP-qPCR、荧光素酶报告等实验结果显示Rfx6可与 IL6启动子区结合并促进IL6的转录；在Rfx6过表达的细胞株中加入IL-6R单克隆抗体后，Rfx6对细胞迁移侵袭和 EMT 的促进作用减弱；体内实验结果提示RFX6促进转移；临床结果显示Rfx6的表达与肝细胞癌患者的临床预后显著相关。.科学意义.在肝细胞癌中Rfx6与肝癌干细胞干性特征相关并在体内外显著促进肝细胞癌细胞转移；这种促癌功能主要通过促进IL6转录激活IL6/STAT3通路实现，Rfx6表达水平与肝细胞癌患者的临床预后显著相关。综上所述Rfx6是潜在的肝细胞癌预后预测分子和治疗靶点。