EZH2表观抑制lnc-RP11D在肿瘤组织间充质干细胞促进胆管细胞癌增殖及转移的作用及机制研究

Cholangiocarcinomas (CCA) are usually characterized by a prominent desmoplastic stroma. Mesenchymal stem cells (MSCs) in tumor tissues can differentiated into cancer-associated fibroblasts when they are interacted with tumor cells. All these phenomena indicate that cancer-asssiciated MSCs may play a pivatal role in CCA progression. Our preliminary results demonstrated that multipotent MSCs existed in CCA tissues and they can promote CCA proliferation in vivo. Next, the mRNA microarray demonstrated that MSCs significantly induced CCA cells epigenetically alteration and primed chromatin state modification. Among those epigenetic genes signigicantly changed, EZH2 expression in CCA cells is significantly upregulated by MSCs and it can sininificantly promote CCA proliferation. Then utilized comprehensive lncRNA microarrays,we analyzed the intersection of lncRNA alteration in CCA cells by MSCs and lentivirus mediated RNA interference in CCA cells (shEZH2) group. We found that lnc-RP11D was significantly repressed by MSC coculture and upregulated by shEZH2 group. The expression of lnc-RP11D is dramatically repressed in CCA tissues compared with corresponding adjacent non-tumor samples by our microarray results and TCGA data. Furthermore, EZH2 overexpression can inhibit the expression of lnc-RP11D in CCA cells. Based on these above results, we proposed the hypothesis that the EZH2-lnc-RP11D axis may play an important role in MSCs modulating CCA proliferation and metastasis. Our research will explore the role and mechanism of how EZH2 epigenetically repressing the expression of lnc-RP11D and investigate which growth factor secreted from MSCs can modulate the axis of EZH2-lnc-RP11D. We hope these findings would provide a new perspective to investigate the interactions between cancer associated MSCs and CCA cells.

胆管细胞癌的组织病理特征为显著的纤维组织异常增生（stroma desmoplasia），肿瘤组织的间充质干细胞（MSC）通常被认为是瘤内成纤维细胞的主要来源之一，提示MSC在胆管细胞癌进程中发挥重要作用。我们预实验表明：1）胆管细胞癌存在MSC且其促进肿瘤增殖和侵袭；2）mRNA芯片显示MSC促进肿瘤表观遗传修饰基因改变，尤其EZH2上调显著和促进肿瘤增殖；3）lncRNA芯片寻找MSC处理的肿瘤细胞和肿瘤中shEZH2共同调控的lncRNA，其中lnc-RP11D在胆管细胞癌显著下调且EZH2抑制lnc-RP11D的表达。我们推测“EZH2表观抑制lnc-RP11D在MSC促进胆管细胞癌进程中发挥重要作用”。本项目将从微环境的视角探讨EZH2表观抑制lnc-RP11D的作用及分子机制，鉴定MSC分泌的因子调控EZH2-lnc-RP11D轴的作用，以期为胆管细胞癌的微环境干预提供新靶点。

肝内胆管细胞癌组织中存在显著的纤维组织异常增生,我们证明胆管细胞癌组织中存在丰富的间充质干细胞（MSC）。被肿瘤激活的MSC显著促进胆管细胞癌的增殖和侵袭能力，lncRNA芯片发现间充质干细胞能够显著促进肿瘤细胞中表观遗传修饰相关的通路发生改变，尤其是EZH2能够显著促进胆管细胞癌和肿瘤干细胞的增殖。进一步通过lncRNA芯片分析，分析MSC调控的lncRNA和RNA干涉EZH2后改变的lncRNA的交集，发现lnc-RP11D可能是MSC和EZH2共同调控的靶lncRNA，其能够显著促进肿瘤细胞增殖。.为了在单细胞水平解析人肝内胆管细胞癌的异质性以及肿瘤微环境与胆管肿瘤细胞之间的相互作用，我们利用5个肝内胆管细胞癌组织和3个癌旁组织进行了单细胞RNA测序分析。一共分析31,302个单细胞，发现10个不同的细胞亚群，包括异常的肿瘤细胞、正常肝细胞、正常胆管细胞、免疫细胞（T细胞、巨噬细胞、NK细胞、B细胞，DC细胞等）、内皮细胞和肿瘤相关成纤维细胞等。因为肝内胆管细胞癌一个显著的病理特征是呈现纤维结缔组织异常增生，因此我们主要关注肿瘤相关成纤维细胞的特征亚群以及与肿瘤细胞的相互作用，通过流式分选富集肿瘤组织中的成纤维细胞进行单细胞RNA测序，发现MCAM阳性的血管样成纤维细胞亚群（Vascular CAF, vCAF）占比最高，且扩增培养的vCAF能够显著促进肝内胆管细胞癌体内外增殖。进一步通过单细胞尺度的生信分析vCAF亚群与肿瘤细胞之间的相互作用，发现vCAF可通过分泌IL-6诱导肿瘤表观遗传改变包括EZH2上调进而促进肿瘤。反过来我们发现ICC肿瘤细胞能够分泌外泌体，且外泌体能富集miR-9-5p，进而上调vCAF中IL-6的表达，反过来促进肿瘤增殖。上述研究结果以通讯作者发表在肝脏领域的顶级期刊Journal of Hepatology和Cancer research,两篇论文均入选ESI高被引研究论文。