脯氨酰顺反异构酶Pin1在肝癌调控微小RNA表达的分子机制研究

Dysregulation of global microRNA（miRNA） expression is discovered in hepatocellular carcinoma (HCC), but the underlying mechanisms remain unclear. Pin1 is the only peptidyl-prolyl cis/trans isomerase that binds to and isomerizes specific phosphorylated Ser/Thr-Pro (pSer/Thr-Pro) motifs in a subset of proteins, resulting in conformational changes in the proteins. Pin1 is overexpressed in HCC. For the first time, we discovered that Pin1 can regulate miRNA expression in HCC cell lines, however, its underlying mechanisms need to be further investigated. Our previous studies showed that XPO5, responsible for pre-miRNA transportation, is phosphorylated and retained in the nucleus in liver tumor, resulting in inability to transport pre-miRNA to the cytoplasm and reduced levels of miRNA expression. Based on our discovery that Ser/Thr phosphorylation residues on XPO5 precedes Proline residue, we hypothesize that Pin1 could regulate miRNA expression through XPO5. The following techniques will be employed in this project, including RNA interference, miRNA microarray, co-immunoprepicipation, site-directed mutagenesis, in vitro isomerization assay, confocal microscopy, RNA-binding protein immunoprecipitation assay, wound healing assay, Transwell cell migration and Matrigel invasion assay. We will study how Pin1 regulates miRNA expression through the change of XPO5 protein conformation and its ability to bind to pre-miRNA, leading to the change of biological function of HCC cells. Furthermore, we will explore the clinical significance of our discovery in HCC patient samples. Therefore, our research will provide experimental and theoretical basis for the treatment of liver cancer using Pin1 as a novel therapeutic target.

miRNA在肝癌中异常表达，但其分子机制仍不清楚。Pin1是蛋白肽基脯氨酰顺反异构酶, 催化磷酸化的丝氨酸/苏氨酸-脯氨酰键发生顺反异构来改变蛋白构象。Pin1的表达在肝癌中显著升高，我们发现Pin1调控miRNA表达，但其调控机制有待研究。我们前期研究表明，负责pre-miRNA转运的XPO5在肝癌中被磷酸化，不能转运pre-miRNA，导致miRNA表达降低。基于我们发现XPO5磷酸化位点Ser/Thr之后是脯氨酸，我们推测Pin1通过XPO5来调节miRNA表达。本项目拟在前期研究的基础上，采用基因芯片、免疫共沉淀、体外异构化分析、细胞迁移和侵袭等方法，从体内外深入研究Pin1如何改变XPO5的蛋白构象及其活性，调控miRNA的表达，进而影响肝癌细胞生物学特性的机制，并探索该发现在肝癌诊断和预测中的临床意义，为Pin1作为新靶点治疗肝癌提供实验依据和理论基础。

肝癌中miRNA生物合成途径的损伤，导致成熟miRNA表达的普遍下调，促进肝癌发生发展。然而，肝癌中miRNA生物合成损伤的分子机制尚不明确。我们前期研究表明，ERK激酶在肝癌中被激活后，磷酸化负责前体miRNA（pre-miRNA）核质转运的XPO5蛋白，使XPO5不能有效地将pre-miRNA从细胞核转运至细胞质，从而下调miRNA表达，促进肝癌演进过程。在此基础上，我们通过生物信息学分析，发现XPO5磷酸化位点（丝氨酸/苏氨酸）之后是脯氨酸。鉴于磷酸化丝氨酸/苏氨酸-脯氨酸基序（pSer/Thr-Pro）是脯氨酰顺反异构酶Pin1的特异性底物，因此我们提出了“Pin1通过调节磷酸化的XPO5（pXPO5）参与miRNA生物合成”的科学假说。. 本项目围绕“肝癌中Pin1介导的miRNA表达调控机制”开展了系统深入的研究，采用高通量测序、免疫共沉淀、体外异构化分析、细胞迁移和侵袭等方法，从分子、细胞和动物层面，阐明Pin1对XPO5蛋白构象及其生物学功能的影响，并在肝癌样本中探索了临床意义。经过四年的研究，取得了以下原创性的科研成果：1）阐明了Pin1识别磷酸化的XPO5，催化其pSer-Pro基序发生顺反异构，降低XPO5与pre-miRNA的结合能力，从而下调肝癌细胞中miRNA表达；2）证实了Pin1可通过影响miRNA表达，调节肝癌细胞的生物学功能，促进肝癌的恶性进展；3）通过计算机辅助药物设计，发现了高活性、高特异性的小分子Pin1抑制剂API-1，显著抑制肝癌发展。这些发现深化了miRNA生物合成调控机制的科学认识，为肝癌的临床诊疗提供了新思路。. 本项目按计划顺利开展全部研究内容，完成各项既定目标，以四川大学为通讯单位在Cancer Cell（IF:23.916）、Hepatology（IF:14.971）、Cell Death & Differentiation（IF:8.086）等国际主流期刊发表论文7篇（均标注项目基金号81572739），其中2篇论文入选“ESI高被引论文”，1篇论文荣获2018年“中华医学百篇优秀论文奖”；同时培养了5名博士，1名硕士。

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