去泛素化酶USP17通过上调KLF4蛋白稳定性促进黑色素瘤适应代谢压力的分子机制研究

Metabolic stress is a common phenomenon inside of melanoma. The adaption to Metabolic stress is vital to melanoma cell survival. Previously, we have discovered that the protein levels of KLF4, a transcription factor, is increased under Metabolic stress, and elevated KLF4 inhibits cell apoptosis, promotes cell growth and metastasis. We then identified deubiquitinase USP17, a member of human DUB family, which could interact with and stabilize KLF4 under Metabolic stress in melanoma cells and found LncRNA H19 was a target gene of KLF4 by sequencing analysis. Based on this, in this project, we will focus on the molecular mechanism by which USP17 regulates KLF4 expression and confirm their relationship as well as the effect of USP17 on the adaption to Metabolic stress via KLF4. Subsequently, we will try to reveal the role of KLF4 in adaption to Metabolic stress and regulation of LncRNA H19 expression. Additionally, we will further prove their effects on melanoma in nude mouse. Finally, we will analyze the association between USP17 , KLF4 and LncRNA H19 as well as the relationship between their expression and the prognosis of patients with melanoma. Taken together, this project will provide novel knowledge and strategy for melanoma therapy.

黑色素瘤作为恶性实体肿瘤，内部常由于葡萄糖缺失形成代谢压力；适应代谢压力，抑制细胞凋亡对黑色素瘤生存至关重要。我们研究发现葡萄糖缺失增加转录因子KLF4表达，阻止其泛素化降解；增加的KLF4抑制黑色素瘤细胞凋亡，促进细胞增殖和转移。随后利用去泛素化酶文库筛选，首次鉴定出USP17作为KLF4去泛素化酶促进其表达；利用测序分析筛选出LncRNA H19为KLF4下游调控基因。基于此，本项目拟研究USP17提高KLF4稳定性的分子机制，明确二者相互作用的关系，并阐明其在黑色素瘤适应代谢压力中的作用；明确KLF4对LncRNA H19调控作用，寻找LncRNA H19下游调控的信号通路，揭示KLF4通过LncRNA H19促进黑色素瘤适应代谢压力的分子机理；并在动物模型中验证三者关系及在黑色素瘤中的作用；最后结合临床资料分析三者相关性及与病人预后的关系；为黑色素瘤治疗提供新思路和方法。

黑色素瘤作为恶性实体肿瘤，内部常由于葡萄糖缺失形成代谢压力；适应代谢压力，抑制细胞凋亡对黑色素瘤生存至关重要。我们在本研究中发现代谢压力诱导KLF4上升，并揭示KLF4下游调控基因。同时我们还发现ITIH5在黑色素瘤中表达下降,低表达ITIH5黑色素瘤病人预后较差；过表达ITIH5抑制黑色素瘤增殖和转移；反之，敲低ITIH5促进黑色素瘤转移和增殖。进一步分子机制研究发现p53转录上调ITIH5表达，并且直接结合在ITIH5启动子。为进一步阐明ITIH5抑制黑色素瘤的分子机制，我们通过免疫共沉淀结合质谱分析，发现ITIH5与KLF4相互作用，并且抑制KLF4转录活性。同时，我们通过去泛素化酶文库筛选发现去泛素化酶USP10是KLF4的去泛素化酶，过表达USP10提高KLF4稳定性，抑制KLF4降解。反之，敲低USP10促进KLF4降解和肺癌形成。在小鼠体内敲低USP10 促进KrasG12D诱导的肺腺癌形成。我们进一步研究发现KLF4转录上调肿瘤抑制因子TIMP3表达，直接结合在TIMP3的启动子上。临床病例分析，发现USP10和KLF4及TIMP3正相关，高表达USP10，KLF4或TIMP3肺癌病人预后较好，生存期增加。最后，我们还发现鸦胆子诱导黑色素瘤中的KLF4下降；过表达KLF4抑制鸦胆子诱导黑色素瘤细胞凋亡。同时我们还发现NCK2作为KLF4下游调控基因参与调控鸦胆子诱导黑色素瘤细胞的凋亡。总之，通过本项目研究我们发现p53 通过调控ITIH5-KLF4轴抑制黑色素瘤的转移和增殖；USP10通过调控KLF4-TIMP3轴抑制肺癌的形成。