QKI结合并调控长链非编码RNA-EGOT影响肝癌细胞增殖和转移的作用机制及临床价值研究

The expression and role of long non-coding RNA EGOT in Hepatocellular carcinoma (HCC) are still unknown. Our preliminary experiments showed that the level of EGOT was significantly higher in tumor tissues than that in paired adjacent non-tumor liver tissues of HCC patients. Up-regulation of EGOT could promote the proliferation and metastasis of HCC cells. We also identified QKI as one of the proteins which could interact with EGOT, and over-expression of QKI could decrease the level of EGOT. Our results also showed that protein A and protein B could interact with both EGOT and QKI. Accordingly, we hypothesized that QKI, protein A and protein B could form complexes, bind to and suppress EGOT to regulate the proliferation and metastasis of HCC cells. In this study, we would thoroughly investigate the roles of the interaction between QKI and EGOT in HCC cell proliferation and metastasis in vitro and in vivo. We would also identify the binding domains from both EGOT and QKI, and analyze how the EGOT-QKI complexes form. Furthermore, we would explore the association of EGOT and QKI levels with HCC occurrence and metastasis by collecting clinical samples of HCC patients. This project will elucidate the roles and clinical values of QKI in HCC progression by interacting with EGOT and regulating its expression, and it will also establish the theoretical basis for the pathogenesis and biomarker in HCC.

长链非编码RNA-EGOT在肝癌中的表达和作用机制尚不明确。我们预实验结果表明EGOT在肝癌肿瘤组织中的表达水平显著高于癌旁组织，过表达EGOT能促进肝癌的生长和转移；其次我们还鉴定出QKI是能和EGOT结合的蛋白，且过表达QKI能下调EGOT水平；另外我们还发现蛋白A和蛋白B既能和EGOT结合又能和QKI结合。为此我们提出科学假设：QKI、蛋白A和蛋白B能形成蛋白复合体结合并下调EGOT水平，并进一步影响肝癌的生长和转移。本课题将在体内、体外条件下继续深入研究QKI结合调控EGOT对肝癌生长和转移的影响；明确EGOT和QKI分子中参与二者结合的分子结构域；阐明EGOT和QKI复合体的构成机制；并研究EGOT、QKI表达水平和肝癌发生、转移的临床相关性。通过以上研究，本课题将阐明QKI结合并调控EGOT在肝癌发生发展中的作用及临床价值，这将为肝癌发病机制及肝癌生物标志物的研究奠定理论基础。

RNA结合蛋白及其调控的非编码RNA在包括肝细胞癌（Hepatocellular carcinoma, HCC）在内的肿瘤发生发展过程中发挥了重要的调控作用。在本课题的研究中，我们通过RNA pull-down、RNA结合蛋白免疫沉淀及蛋白质谱等技术，明确了HCC细胞中RNA结合蛋白QKI能够结合并调控长链非编码RNA EGOT的表达，QKI蛋白的KH、QUA2结构域以及EGOT的5’端序列能够参与二者的结合，我们还鉴定出QKI-EGOT复合体中另外两种组成蛋白；细胞和动物实验结果表明，EGOT和QKI均能够促进HCC细胞的增殖、克隆形成、迁移、侵袭以及小鼠成瘤能力；为了进一步探究EGOT和QKI的临床意义，我们收集HCC患者的肿瘤组织并进行PCR检测，实验结果表明EGOT和QKI在HCC肿瘤组织中的表达升高并且和HCC患者的不良预后相关。以上研究结果明确了QKI及EGOT在HCC发生发展中的作用及临床价值，为探寻新的HCC生物标志物和治疗靶点提供理论基础。同时，非酒精性脂肪性肝病（Non-alcoholic fatty liver disease, NAFLD）及酒精性肝病（alcoholic liver disease, ALD）是HCC的重要病因。我们利用磷脂酰丝氨酸ELISA及基因敲除小鼠构建等技术，揭示了NAFLD患者肝组织DRAM1的表达水平以及血浆外泌体水平升高，脂肪酸诱导的DRAM1表达升高能够通过结合STOM蛋白影响溶酶体膜透化进而促进肝细胞外泌体分泌；通过腺相关病毒的制备及蛋白免疫共沉淀等技术，我们发现酒精诱导的肝细胞DRAM1表达升高能够增加细胞外囊泡中PKM2蛋白水平进而促进巨噬细胞炎症因子的表达并加剧ALD的疾病进程。通过我们的研究，能够为探寻新的NAFLD和ALD的诊断标志物和治疗靶标提供理论基础，对提高HCC防治水平有积极意义。