肝刺激因子通过双特异性磷酸酶2调控肝癌细胞转移的研究

Currently the effect of hepatocellular carcinoma (HCC) treatment remains to be improved. And there are needs to search for more detailed molecular mechanism for HCC pathogenesis and to find new therapeutic targets. We found that the expression levels of hepatic stimulator substance (HSS) were low in tumors among patients with HCC metastasis. And the cells with low expression levels of HSS had increased ability of migration, indicating that the down regulation of HSS is correlated to HCC metastasis. However, its molecular mechanism remains to be understood. Our previous studies revealed HSS down-regulation resulted in ERK activation. The expression levels of a specific ERK inhibitor, dual-specificity phosphatase (DUSP) 2 decreased in the cells with low expression levels of HSS. And the expression of HSS and DUSP2 are closely correlated in HCC. We hypothesize that the down regulation of HSS causes a decrease in DUSP2, which resulted in sequential alterations including ERK activation, epithelial-mesenchymal transition, and HCC metastasis. In our current study, research is going to be conducted with cell model as well as animal model, using techniques including Western blot, siRNA, cell migration assay, high content analysis, reporter gene analysis, and chromatin immunoprecipitation, and injection into nude mice to investigate the mechanism of the DUSP2 down-regulation. Human HCC tissue chip would also be used to confirm this finding. We hope this project would help us to find a new pathway related to HSS metastasis and to provide new potential therapeutic targets for HCC treatment.

目前肝癌分子治疗效果不佳，尚需深入了解肝癌进展分子机制以获得新的治疗靶点。我们发现肝刺激因子（HSS）在肝癌转移患者肿瘤组织内异常低表达，且HSS低表达的细胞迁移能力增强，提示HSS表达下调与肝癌转移相关，但其中的分子机制尚不明了。前期发现HSS表达下调后ERK异常活化，而ERK的特异性抑制剂，双特异性磷酸酶2（DUSP2）表达下降，肝癌组织内DUSP2表达与HSS表达密切相关。据此提出假设，HSS表达下调使DUSP2表达下降，ERK异常活化，诱导上皮-间质转化，促进肝癌转移。本课题拟在细胞及动物水平，利用Western blot、siRNA、细胞迁移实验、高内涵细胞成像分析技术及裸鼠体内注射等方法阐明此假说，并通过报告基因分析及染色质免疫沉淀等实验确定DUSP2表达下降的调控机制；最后应用人肝癌组织芯片加以证实。此研究将建立新的与肝癌转移相关的分子通路，为肝癌分子治疗提供新的潜在靶点。

目前肝癌分子治疗效果不佳，尚需深入了解肝癌进展分子机制以获得新的治疗靶点。我们发现肝刺激因子（HSS）在肝癌转移患者肿瘤组织内异常低表达，且HSS低表达的细胞迁移能力增强，提示HSS表达下调与肝癌转移相关，但其中的分子机制尚不明了。本课题首先利用Western blot方法发现HSS表达下调使肝癌细胞内上皮细胞分子标志物表达降低，间质细胞分子标志物表达上调，ERK磷酸化水平异常升高。裸鼠脾脏注射肝内转移实验结果显示，注射HSS低表达肝癌细胞的裸鼠肝脏内转移瘤更多，而且死亡时间早于注射对照细胞裸鼠。随后应用透射电镜和Real-time PCR等实验检测线粒体形态和生物生成分子标志物表达的变化，结果显示HSS低表达使细胞内线粒体数目明显增多，形状变圆。HSS低表达细胞内线粒体融合相关基因Mfn2的表达和mtDNA的相对含量明显降低，与mtDNA复制、转录相关的PGC-1α和TFAM的表达及由mtDNA转录并翻译的ATP合酶（ATPs）和细胞色素c氧化酶IV（Cox-IV）的表达也随之降低；与之对应ATPs和Cox-IV的活性减弱，细胞内ATP含量显著降低。肝星状细胞（HSCs）作为一种重要的肝基质细胞，其活化可以促进肝癌细胞的侵润和转移。我们在前期实验中发现HSS低表达使HSCs线粒体钙离子浓度增加促进HSCs活化。为研究其中的分子机制，本课题应用免疫共沉淀等实验发现HSS和Mia40存在相互作用，而且HSS低表达使细胞内还原型Mia40增多。转染Mia40后用Rhod-2标记线粒体钙离子，流式细胞仪检测结果显示Mia40使HSS低表达细胞钙离子含量下降，Transwell实验结果显示Mia40降低了HSS低表达细胞的迁移能力。本课题探讨了HSS调节不同细胞迁移的分子机制，并寻找到了HSS的直接分子靶点，为全面认识这一重要基因在肝癌中的作用提供了新的理论依据。