低氧条件下OGT/OGA介导的FBP1糖基化修饰对胰腺癌进展的机制研究

Fructose -1,6 biphosphatase 1 (FBP1) is an important rate-limiting enzyme in gluconeogenesis.However, the function of FBP1 as tumor suppressor and its relationship with pancreatic cancer are still unclear. Our previous study suggested that the low expression of FBP1 in pancreatic cancer was associated with a poor prognosis and mass spectrum result indicated FBP1 may interact with the glycosylation enzyme OGT/ deglycosylase OGA. Therefore, we propose the hypothesis that hypoxia can induce the interaction change between OGT/OGA and FBP1 in pancreatic cancer cells, which promotes FBP1 glycosylation and degradation/inactivation, leads to increase the tumor metabolic rate and ultimately acceleratesthe progression of pancreatic cancer. In order to verify this hypothesis, we used pancreatic cancer cell lines and tissues to explore the important role of FBP1 in the progression of pancreatic cancer from the aspects of molecular, cell and tissue and animal levels. Besides, we also want to figure out the underlying mechanism of FBP1 protein function change regulated by glycosylation modification induced by OGT/OGA under hypoxia condition. This study will reveal new mechanisms of pancreatic cancer metabolism, which could provide new ideas about metabolic therapeutic targets in the future treatment of pancreatic cancer.

果糖-1,6 -二磷酸酶1（FBP1）是糖异生途径中的重要限速酶，但其作为肿瘤抑制因子与胰腺癌进展的关系尚不清楚。我们前期研究发现胰腺癌中FBP1低表达提示不良预后，且质谱结果提示FBP1可以与糖基化酶OGT/去糖基化酶OGA相互作用。目前尚没有FBP1糖基化修饰功能研究的报道，为此，我们提出假设，胰腺癌中的缺氧环境可以诱导OGT/OGA与FBP1作用模式发生变化，促进FBP1糖基化修饰并发生降解/功能失活，导致肿瘤代谢速率增加，最终加快胰腺癌进展。为了验证该假说，我们将通过胰腺癌细胞系及胰腺癌组织，从分子、细胞、组织以及动物整体水平等多方面探讨FBP1在胰腺癌进展中的重要作用，明确低氧条件下OGT/OGA通过诱导FBP1糖基化修饰以及对FBP1蛋白功能的调控机制。本研究将从FBP1糖基化这个新视点揭示胰腺癌肿瘤代谢新机制，为未来代谢靶向治疗提供新思路。

组蛋白去乙酰化酶SIRT7在胰腺癌组织内普遍表达量升高，且高表达组较低表达组预后更差，提示可能与肿瘤发生发展相关。免疫共沉淀结合质谱结果SIRT7可以与糖基化转运酶OGT相互作用，后续免疫共沉淀实验与免疫荧光实验验证了该结果。接着，我们为了探究糖基化对SIRT7的作用，我们验证了糖基化好后内源性SIRT7的表达量，结果提示糖基化可以在蛋白质水平促进其表达，且这种表达是通过抑制蛋白酶体介导的泛素化降解来实现的。同时，SIRT7糖基化后可以促使其介导的H3K18Ac水平减弱，其下游共同调控的靶基因表达量会发生变化，转录因子的富集程度也会改变。我们通过质谱的方式确定SIRT7发生糖基化修饰的位点位于377位的丝氨酸。将其点突变为丙氨酸可以发现其调控的蛋白质糖基化水平减弱。将野生型SIRT7与突变型SIRT7比较后可以发现，剥夺其糖基化能力后其泛素化水平可恢复。以上结果是首次报道的SIRT7糖基化修饰的结果。.FBP1蛋白在胰腺癌组织中表达量下降，而胰腺癌组织中糖基化水平下降，免疫共沉淀已经证实FBP1可以被糖基化，其糖基化位点位于第46位的丝氨酸。但是糖基化之后，FBP1的蛋白质水平并没有发生差异，新的机制有待探索。