靶向肝星状细胞SYK抑制肝癌发生的分子机制与功能研究

Activation of hepatic stellate cell (HSC) plays a key role in liver fibrogenesis. Recently, the effect of HSC activation on the tumorigenesis and progression of hepatocellular carcinoma (HCC) has been increasingly studied, but the underlying mechanism remains not well characterized. In our previous study, we have reported SYK-regulated tumor invasion and metastasis through the differential expression of two SYK isoforms in hepatocellular carcinoma. We sought to further investigate the role of SYK in HSCs, which constitute a major subtype of stromal cells in the liver. We found that SYK protein levels in liver tissues increased with hepatitis viral (HBV or HCV) infection, which occurred mainly in HSCs. Furthermore, HSC activation was positively associated with SYK upregulation. Importantly, SYK-targeted inhibitor significantly reduced HSC activation, liver fibrosis and the formation of hepatocellular carcinoma in vitro and in vivo. We therefore hypothesized that SYK inhibitor as an effective therapy to attenuate liver fibrosis and development of hepatocellular carcinoma by inhibiting activation of HSCs. To investigate the impact of SYK inhibition on HSC activation and its biological effects, we propose to use SYK-targeted anti-fibrosis treatment in several murine liver fibrosis/HCC models. Furthmore, we will apply mass spectrometry combined with bioinformatics analysis, chromatin immunopreciptation (ChIP), RNA-sequencing and other technologies to explore the molecular mechanism of SYK-mediated HSC activation. In addition, we plan to measure SYK signaling pathway protein expression in human liver samples to explore SYK with the risk of hepatocellular carcinoma tumorigenesis. The study in this grant proposal will help to elucidate the mechanism of SYK-mediated HSC activation and the biological effects of SYK targeted therapy, which could be significant in clinical and translational settings.

肝星状细胞(HSC)活化是肝纤维化发生的中心环节，且在肝癌发生发展中的作用日益明确，但机制仍不明。我们前期研究已揭示：肝癌细胞中SYK基因可通过其异构体的差异表达，参与调控肿瘤的侵袭转移。最近我们对HSC中SYK的研究取得重要进展：发现HBV或HCV感染的肝组织中SYK表达均明显升高，且主要发生在其间质细胞HSC中；HSC的活化程度与其SYK表达呈正相关；靶向SYK可显著抑制HSC活化、肝纤维化及肝癌形成。因此，推测SYK可能在肝癌早期的肝纤维化、肿瘤形成中发挥重要作用。本项目拟采用多种动物的肝纤维化/肝癌模型，研究SYK对HSC活化及其生物学效应的影响；利用质谱、ChIP、RNA测序等技术方法，探讨SYK促进HSC活化的分子机制；通过在人肝组织中的验证，明确SYK相关的肝癌发病风险。本研究旨在阐明SYK促进HSC活化的分子机制，及靶向SYK治疗的生物学效应，具有重要的临床转化应用价值。

脾酪氨酸激酶（Spleen tyrosine kinase, SYK）在免疫细胞信号通路中发挥重要作用，且在人肝细胞癌中被报道为一种新的生物标志物。本项目旨在研究SYK促进肝纤维化的机制，并评价SYK作为肝纤维化和肝癌发生发展的治疗靶点。..本项目分析了SYK在肝脏细胞中的细胞表达定位情况，以及在36例正常、127例HBV和22例HCV感染的人肝组织中SYK表达水平与与肝纤维化程度的相关性；使用PCR阵列检测SYK敲低后肝星状细胞中转录因子表达的改变。在人肝星状细胞系（LX-2和TWNT-4）、人原代肝星状细胞以及多种动物模型中开展SYK抑制剂GS-9973的抗肝纤维化/肝癌等体内外实验，动物模型包括四氯化碳诱导的小鼠肝纤维化模型、二乙基亚硝胺或胆管结扎诱导的大鼠进展性纤维化/肝硬化动物模型，其中二乙基亚硝胺大鼠模型可伴有肝癌自发形成。我们分别从现象、机制与靶向治疗三个层次开展研究，探讨SYK促进肝星状细胞激活与肝纤维化的机制，并评价SYK抑制剂GS-9973的体内外抗肝纤维化与肝癌发生的疗效。..研究结果提示：在人肝组织中，肝星状细胞和肝细胞中的SYK蛋白与肝纤维化/肝硬化分期呈正相关；HBV或HCV感染显著增加肝细胞SYK和细胞因子的表达；引起肝细胞中细胞因子分泌增多，可进一步诱导肝星状细胞中SYK及肝纤维化相关基因的表达；肝星状细胞SYK的表达上调，通过增加与肝星状细胞激活相关的特异性转录因子的表达，从而促进肝星状细胞激活与增殖。SYK特异性拮抗剂GS-9973可有效抑制SYK激酶活性，通过抑制星状细胞的激活，有效抑制肝纤维化进程，降低梗阻性黄疸，并抑制动物模型HCC的发生。..结论：SYK通过促进肝星状细胞激活加剧肝纤维化，SYK是一种肝纤维化和预防肝癌发生的有效潜在干预靶点。