FBP1负向调控上皮间质转化在肝细胞癌侵袭转移中的作用及其机制研究

Rewiring of energy metabolism plays an important role in the pathogenesis of hepatocellular carcinoma(HCC). Our previous study proved that a key enzyme in gluconeogenesis, fructose-1,6-biphosphotase-1 was downregluated in HCC tissue, FBP1 was an independent prognostic factor of overall and recurrence-free survival, furthermore, FBP1 inhibited HCC proliferation. Epithelial-mesenchymal transition(EMT) is a critical process for tumor invasion and metastases. By analysis of TCGA database, we found FBP1 expression in HCC tissue was lower in patients who developed distant metastases than those who didn't, FBP1 expression negatively correlated with expression of EMT signalling molecules. We suppose FBP1 also modulates EMT in HCC. We plan to over-express or knockdown FBP1 in HCC cell lines, exploiting in vitro cell culture/in vivo animal models to study the role of FBP1 in modulating EMT of HCC. Furthermore, we'll perform luciferase assay, co-immunoprecipitation assay, chromatin immunoprecipitation assay to study whether FBP1 affects the key EMT transcription factor Snail and Twist1 to form gene silencing complex and their ability to bind E-Box of target genes. The study will enrich our knowledge of carbohydrate-metabolizing enzymes, and lay a ground for future researches.

能量代谢异常是肝细胞癌（HCC）的重要发生机制。我们前期研究证实：果糖-1,6-二磷酸酶-1（FBP1）作为糖异生限速酶在HCC癌组织表达下调，FBP1是术后总生存和无复发生存的独立预测因子，FBP1抑制了HCC细胞增殖。上皮间质转化（EMT）是肿瘤发生侵袭、远处转移的主要机制。课题组利用TCGA数据库分析发现，发生术后转移的HCC癌组织FBP1表达较低，FBP1与EMT信号通路分子表达负相关 ，我们推测FBP1也可能对HCC的EMT有重要影响。本课题旨在前期研究基础上，在HCC细胞系中过表达或敲除FBP1，通过体外细胞实验和体内裸鼠成瘤实验探索FBP1对HCC EMT的调控。还将利用荧光素报告实验、免疫共沉淀、染色质免疫共沉淀，研究FBP1是否影响EMT关键转录因子Snail、Twist1基因沉默复合物形成以及Snail、Twist1与靶基因E-Box的结合能力，明确FBP1作用靶点。

能量代谢异常是包括肝细胞癌在内恶性肿瘤的重要特点，表现为糖酵解增加，而与之对应的糖异生受到抑制。果糖-1,6-二磷酸酶-1（FBP1）是糖异生限速酶之一，我们此前的研究已发现FBP1在肝癌细胞系和肝癌组织里表达下调，且下调的程度与肿瘤侵袭增殖、肝癌患者的预后密切相关，但FBP1在肝癌中表达下调的机制仍不明确。曾有报道启动子甲基化是FBP1在部分肿瘤表达下调的原因。通过不同的方式检测后，并未发现肝癌组织FBP1启动子存在广泛甲基化。进一步生物信息学分析发现，miR-21-3p可能结合于FBP1 mRNA的3'端非编码区，TCGA等数据库分析表明，肝癌组织中miR-21-3p表达明显上调，且与FBP1表达和患者预后负相关。荧光素报告实验和随后的过表达实验证实miR-21-3p靶向FBP1。细胞系和裸鼠成瘤实验表明，FBP1抑制了肝癌细胞系的增殖和侵袭能力，而过表达miR-21-3p则显著促进了肝癌细胞系的增殖和侵袭，并且miR-21-3p的这种能力可以被共转染FBP1所抵消。由此可见，miR-21-3p是肝细胞癌负调控糖异生的重要因子，为治疗肝癌提供了新的途径。