肝星状细胞microRNA-199在肝硬化及肝癌发生中的作用和机制研究

The activation of hepatic stellate cells (HSCs) is a key process in liver cirrhosis and carcinogenesis of hepatocellular carcinoma (HCC). Molecular regulatory mechanisms of hepatic stellate cells activation is one of the crucial topics in scientific research of liver cirrhosis and HCC carcinogenesis. microRNAs play vital roles in carcinogenesis and progression of HCC. Previously, we have analyzed the miRNomes of human normal liver, hepatitis liver, cirrhotic liver, and HCC specimen, and found that miR-199 expression was enriched in human normal liver and plays a significant role in HCC progression. The study published in Cancer Cell, 2011. However, the roles of miR-199 in HSCs in the promotion of liver cirrosis and HCC carcinogenesis remain elusive. Hence, the miR-199 hepatic stellate cell specific knockout mice and miR-199 hepatocyte specific knockout mice were constructed, respectively, and found that HSC specific deficiency of miR-199 significantly decreased HCC carcinogenesis and alleviated liver cirrhosis in the CCl4-induced HCC carcinogenesis mouse model. Thus, we would like to clarify the function and molecular mechanism of activation of HSCs in promoting liver cirrosis and HCC carcinogenesis regulateded by miR-199, and thus lay the foundation for screening novel molecular targets for the treatment of HCC and facilitating the development of effective therapeutics against HCC.

肝星状细胞活化是肝硬化及肝癌发生的关键环节，肝星状细胞活化调控的分子机制研究是本领域的重大科学问题。microRNA在肝癌发生发展中发挥关键调控作用。本课题组前期工作揭示了肝癌、肝炎、肝硬化及正常肝组织的microRNA组，发现正常肝脏中富集表达的miR-199在肝癌进展中发挥了重要作用，本工作发表于Cancer Cell, 2011。然而，miR-199在肝脏星状细胞中的表达及其在肝硬化及肝癌发生中的作用目前未知。我们发现在肝硬化和肝癌发生疾病进展中，肝星状细胞miR-199表达显著增高。据此，我们构建了miR-199的肝星状细胞敲除小鼠模型，在CCl4诱导小鼠肝硬化及肝癌发生模型中，miR-199肝星状细胞敲除的小鼠肝硬化程度明显减轻，肝癌发生显著降低，由此，本研究拟探求肝星状细胞中miR-199在调控肝硬化及肝癌发生中的作用和机制，为肝癌发生的早期干预提供潜在靶点。

在本研究中我们探索了miR-210在促肝细胞癌新生血管生成中的相关功能及机制研究。我们首先比较分析了正常肝组织、肝炎肝组织、肝硬化肝组织、癌旁组织及不同分期肝癌组织中的miR-210的表达差异情况，发现随着疾病的进展，miR-210的表达不断上调，并且其表达水平和HCC的临床分期、肿瘤进展、复发及转移密切相关。随后我们分析比较了miR-210的表达水平和肝细胞癌患者临床病理特征之间的关系，发现miR-210高表达的患者其肿瘤直径更大，分化程度更低，且合并血管侵犯及无完整肿瘤包膜的比例更高，提示miR-210高表达患者其肿瘤更具侵袭性，单因素及多因素分析均表明miR-210的表达水平是影响肝细胞癌患者行肝切除术后复发及生存的独立危险因素，生存分析也表明，miR-210高表达患者其无瘤生存率及总体生存率均显著低于miR-210低表达患者。细胞学实验表明，miR-210表达升高可以明显促进新生血管生成，免疫组化实验分析表面在miR-210高表达患者的HCC组织中，CD34表达也明显升高，两者表达情况呈正相关，裸鼠皮下成瘤实验表明过表达miR-210后，裸鼠皮下肿瘤中CD34的表达情况显著高于对照，表明miR-210可以促进HCC新生血管生成。进一步的机制实验表明miR-210可以通过下游的靶基因FGFRL1发挥其促血管生成功能。