蛋白磷酸酶PP2A在肝癌调控微小RNA表达的分子机制研究

miRNA在肝癌中异常表达，但其分子机制尚未阐明。由XPO5负责的pre-miRNA的核质转运是miRNA生物合成的一个关键步骤。我们前期研究表明，XPO5在肝癌中被ERK激酶磷酸化后，被脯氨酰顺反异构酶Pin1识别并使其蛋白构象发生改变，因此，XPO5不能有效转运pre-miRNAs，导致肝癌中成熟miRNA表达降低。蛋白磷酸化修饰是一个可逆的过程。基于我们发现蛋白磷酸酶PP2A可调控XPO5的磷酸化水平，我们推测PP2A通过XPO5调节miRNA表达，但其具体的分子机制有待研究。本项目拟采用高通量测序、免疫共沉淀、RNA pull-down分析、免疫荧光、细胞迁移和侵袭等方法，从体内外深入研究PP2A如何改变XPO5的磷酸化水平及其活性，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PP2A作为肝癌治疗新靶点提供实验依据和理论基础。

Downregulation of global microRNA（miRNA） expression is discovered in hepatocellular carcinoma (HCC), but the underlying mechanism remains elusive. The pre-miRNA transportation from the nucleus to the cytoplasm by XPO5 is a critical step for miRNA biogenesis. We have demonstrated that XPO5 is phosphorylated by ERK kinase, and subsequently recognized and isomerized by peptidyl-prolyl cis/trans isomerase Pin1, so that pre-miRNAs can not be transported and retain in the the nucleus, leading to the decreased expression of mature miRNA in HCC. Protein phosphorylation and dephosphorylation are reversible processes, which are important for regulation of protein function. Based on our discoveries that XPO5 phosphorylation sites are Ser/Thr and the protein phosphatase PP2A can dephosphorylate XPO5, we hypothesize that PP2A regulates miRNA expression through dephosphorylation of XPO5. However，the detailed mechanism needs further investigation. In this study, we will clarify how PP2A regulates miRNA expression in HCC through its effect on XPO5 phosphorylation and its nucleo-to-cytoplasmic transport ability of pre-miRNAs by taking advantage of the following techniques, high-throughput sequencing of small RNAs, co-immunoprecipitation, RNA pull-down assay, immunofluorescence, wound healing assay, Transwell cell migration and Matrigel invasion assay. 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 PP2A as a novel therapeutic target.