ECD通过抑制E3连接酶ZFP91对hnRNPF蛋白的泛素化降解促使肝癌细胞侵袭转移的分子机制研究

The most critical cause for hepatocellular carcinoma-related death is cancer metastasis. Our previous papers indicated that ECD is a key downstream effector of ACK1 in the induction of EMT and metastasis. Although ECD is required for cell-autonomous roles in development and oogenesis in Drosophila, the functional roles of ECD in cancer progression and metastasis is still unknown. To discover the cue of ECD to induce cancer invasion and metastasis, a known protein hnRNP F-associated with cancer metastasis is preliminarily found to be interacted with ECD by using co-immunoprecipitation together with proteomics technology. And our preliminary experiments showed that ECD up-regulated the hnRNP F protein level, instead of the hnRNP F mRNA level, suggesting that ECD may increase the stability of hnRNP F protein. We further demonstrate that E3 ligase ZFP91 possibly interacts with hnRNP F, suggesting that ZFP91 may induce the ubiquitination of hnRNP F proteins.And we further found that ECD interacts with ligase E3 ZFP91.Therefore, a scientific hypothesis in which ECD inhibits E3 ligase ZFP91-induced hnRNP F protein ubiquitination degradation to stimulate cancer cell EMT and metastasis by interacting with ZFP91 is here provided..In the project, based on the novel findings above, the correlations between the expression of ECD and clinico-pathological features will be investigated, and the correlation of ECD with a poor prognosis in patients with hepatocellular carcinoma will be analyzed. The project will demonstrate that ECD stimulates hepatocellular carcinoma EMT and metastasis by stabilizing hnRNP F protein in vivo and in vitro. E3 ubiquitin ligase ZFP91-mediated hnRNP F degradation will be discovered and validated, the regulation ubiquitin sites in hnRNP F protein which are ubiquitin-regulated by the E3 ubiquitin ligase ZFP91 will discovered and validated. The molecular mechanism in which ECD blocks the interaction of ZFP91 with hnRNP F by competitively interacting with ZFP91, ECD inhibits the hnRNP F ubiquitination and degradation, and then ECD up-regulates the hnRNP F protein level, will be fully elucidated in the project. .In conclusion, the functional roles and molecular mechanism of ECD in hepatocellular carcinoma metastasis will be fully elucidated in the project. The study will be helpful for us to discover novel progonsis biomarkers and drug targets for intervening hepatocellular carcinoma invasion and metastasis, and broaden and enrich our view about cancer invasion and metastasis.

侵袭转移是肝癌致死的关键原因。我们前期研究发现ECD是ACK1促使肿瘤侵袭转移的一个关键效应基因。但ECD如何调控肿瘤侵袭转移还没有报道。在前期预实验中，我们在细胞模型中发现ECD促使肝癌侵袭转移；ECD与hnRNP F蛋白可能存在相互作用，并上调其蛋白而不是mRNA水平，提示ECD可能增强了hnRNP F蛋白稳定性；进一步发现ZFP91可能是调控hnRNP F蛋白泛素化的E3连接酶，并且ECD与ZFP91也存在相互作用。据此我们提出科学假说：ECD通过与E3连接酶ZFP91结合，抑制ZFP91对hnRNP F蛋白的泛素化降解，而上调hnRNP F蛋白水平，从而促使肝癌侵袭转移。本项目拟在临床样品、细胞模型和动物实验中，回答ECD通过抑制ZFP91对hnRNP F蛋白泛素化降解，促使肝癌侵袭转移的科学问题。阐明ECD调控肿瘤侵袭转移的分子机制，为发现肝癌预后标记和药物干预靶标提供理论依据。

肝癌的转移是肝癌致死的关键原因，但肝癌侵袭转移的机制目前还不是很明确。ECD作为一个癌基因ACK1的重要效应基因，其在肿瘤中的功能机制尚不明确。我们发现，ECD在肝癌中上调，ECD的表达上调与肝癌的侵袭转移等恶性表型密切相关，其可以作用肝癌预后差的一个独立潜在预后标志物。证实ECD促使了肝癌的侵袭转移等生物学功能。阐明了ECD通过竞争性结合E3泛素连接酶ZFP91，阻断了ZFP91对hnRNP F和hnRNP K等癌蛋白的泛素化和降解，导致了hnRNP F和hnRNP K蛋白水平在肝癌等恶性肿瘤中上调，从而促使了肝癌等恶性肿瘤的侵袭转移。发现了调控hnRNP F和hnRNP K蛋白泛素化修饰的E3连接酶ZFP91。阐明了ZFP91通过泛素化hnRNP A蛋白调控PKM基因pre-mRNA剪切，抑制肝癌细胞代谢重编程，从而抑制肝癌发生发展和侵袭转移的功能机制。