CSN5调控HK2介导糖酵解途径促进肝癌侵袭转移的机制研究

The abnormal activity of glycolytic metabolism mediated by hexokinase 2 (HK2) is closely related to the invasion and metastasis of hepatocellular carcinoma (HCC), but the mechanism of HK2 regulation has not been fully elucidated. Our previous study have confirmed that CSN5 can promote the invasion and metastasis of HCC. Further preliminary studies suggest that CSN5 and HK2 were up-regulated and positively correlated in HCC; down-regulation of CSN5 could inhibit the expression of HK2 in HCC cells, and the results of intracellular glycolytic enzyme activity show that CSN5 silence resulted in the decrease of LDH activity in HCC cells and the reduction of lactic acid concentration in cell supernatant; In addition, CSN5 was directly bound to HK2 in HCC cells by protein mass spectrometry and immunoprecipitation assay; In combination with the relevant literature and CSN5 silenc could lead to an increase in the ubiquitination level of HK2, we hypothesize that CSN5 stabilizes HK2 expression via inhibiting its ubiquitination level, and then enhances glycolytic pathway, leading to promote the invasion and metastasis of HCC. To test this hypothesis, we will construct stable transfected cell lines and nude mice transplantation tumor model, to investigate the effect of CSN5 on the invasion and metastasis of HCC by regulating glycolytic pathway. We further demonstrate the mechanism through which CSN5 enhances glycolytic pathway by regulating HK2 expression by in vitro ubiquitination experiments. This study will provide a new theoretical basis for clarifying the mechanism of invasion and metastasis of HCC.

己糖激酶2（HK2）介导的糖酵解代谢异常活跃与肝癌侵袭转移密切相关，但HK2被调控机制尚未完全阐明。我们前期研究证实CSN5可促进肝癌侵袭转移。进一步预实验发现：肝癌组织中CSN5及HK2蛋白均高表达，且呈正相关；下调CSN5可降低肝癌细胞中HK2的表达，胞内糖酵解酶活性检测结果显示沉默CSN5导致肝癌细胞胞液LDH活性减弱，细胞上清液乳酸浓度下降；另外，质谱鉴定和免疫共沉淀结果表明，CSN5与HK2直接结合。结合相关文献并发现沉默CSN5可导致HK2的泛素化水平增加后，我们推测：CSN5抑制HK2的泛素化水平稳定其表达进而增强糖酵解途径，促进肝癌侵袭转移。为验证假设，本研究拟先构建稳定转染细胞株及裸鼠肿瘤模型，明确CSN5通过调控糖酵解途径对肝癌侵袭转移的影响；再利用体外泛素化实验等技术探讨CSN5调控HK2增强糖酵解途径的具体机制。本研究结果将为阐明肝癌侵袭转移机制提供新的理论依据。

糖酵解代谢异常活跃与肝癌侵袭转移密切相关。研究表明己糖激酶2（HK2）是调控肝癌糖酵解途径的重要效应分子，但是其被调控机制尚未完全阐明。本团队既往研究证实CSN5可促进肝癌侵袭转移，本项目研究进一步发现：①下调肝癌细胞中CSN5表达后，肝癌细胞糖酵解代谢水平显著下降；②CSN5通过HK2调控肝癌细胞糖酵解；③CSN5通过HK2介导的糖酵解促进肝癌侵袭和转移；④在肝癌细胞中，CSN5通过抑制HK2泛素化降解进而稳定其表达；⑤CSN5蛋白与HK2蛋白存在相互作用；⑥姜黄素抑制肝癌细胞糖酵解水平，并减少肿瘤细胞侵袭转移能力。本研究结果为阐明肝癌侵袭转移机制提供新的理论依据以及临床转化前景。