PGC-1α介导的miRNA前体选择性加工在血糖稳定调节中分子机制研究

PGC-1α regulates glucose homeostasis as a crucial co-activator through its expression and post-translational modification responding to fasting-feeding cycle. Our previous data indicated that PGC-1α represented a fine-tuning regulatory role in hepatic gluconeogenesis via a miR-29-mediated negative feedback network motif. However, several questions remained unanswered. For example, whether PGC-1α contributing to glucose homeostasis via miRNA-mediated genetic pathways is a global mechanism? Does PGC-1α hold novel function in cytoplasm after protein modification? Does PGC-1α function as an RNA binding protein in the processing of miRNA precursor? In this project, we aimed to answer these questions. Firstly, RNA sequencing and metabolic phenotypes analysis will be employed to define the global mechanism of PGC-1α/miRNA-mediated network motif in regulating glucose homeostasis. Secondly, we seek to indicate the novel function of inactive PGC-1α in the cytoplasm via investigating the difference between the proteins complex of nucleic and cytoplasmic PGC-1α by using an improved two-hybrid system. The last and most important, we hope to elucidate the function of PGC-1α as an RNA binding protein in the cytoplasm and clarify the molecular mechanism of PGC-1α modifying glucose homeostasis via regulating the processing of miRNA precursor. Altogether, we attempt to elucidate the regulatory mechanism of PGC-1α-mediated miRNA processing in cytoplasm and clarify the novel function of inactive PGC-1α in the maintenance of blood glucose level during fasting-feeding cycle via investigating the interaction among gene layers in the genome. We also want to validate novel therapeutic target for type II diabetes through this project.

PGC-1α是一个多功能的转录共激活因子，在机体代谢调节中发挥重要功能。申请者前期工作显示，PGC-1α-miR-29调控网络可精细调节机体的血糖水平。但是，仍有几个关键问题尚未解决。如PGC-1α通过介导miRNA参与糖代谢调控是否是广泛的机制？翻译后修饰导致PGC-1α细胞定位改变是否具有功能？PGC-1α是否做为RNA结合蛋白参与miRNA前体选择性加工，并以此方式调控糖代谢？申请者拟通过RNA测序结合特异miRNA的代谢表型分析，揭示PGC-1α-miRNA调控网路参与血糖稳态调节的通用机制；通过酵母双杂交技术，解析胞质与胞核PGC-1α蛋白质复合物并阐述胞质内“失活”PGC-1α的新功能；研究PGC-1α做为RNA结合蛋白调控miRNA前体的选择性加工，并以此方式影响机体血糖稳态调节的作用机制。本课题将从基因信息层互作层面揭示血糖稳态调控的分子基础，为2型糖尿病治疗提供新的靶点。

PGC-1α主要定位于细胞核，通过自身表达调控及翻译后修饰的改变对饮食周期产生应答，参与机体血糖的稳态调节。本项目拟阐述胞质内“失活”PGC-1α的新功能；研究PGC-1α做为RNA结合蛋白参与miRNA前体的选择性加工调控，并以此做为分子基础，调控机体糖代谢的作用机制。课题采用蛋白质互作与蛋白质-RNA互作分析方法未发现可受PGC-1α调控的特异miRNA。因此，本研究将重点转向参与肿瘤细胞糖代谢调节的miR-449家族，结果显示，miR-449在肝癌组织中表达下调，恢复其表达可以抑制肝癌细胞的沃伯格效应，介导糖酵解/有氧氧化转换。分子机制研究则显示，miR-449可以直接调控ERRA-PGC-1β-LDHA转录调控轴，上述三个分子为miR-449的直接功能靶基因。全基因组甲基化分析显示，启动子区域甲基化诱导miR-449失活，去甲基化药物则可以激活miR-449并抑制肝癌细胞糖酵解，提示去甲基化类小分子或可作为表观药物用于肝癌治疗。后续在CDX（Cell-derived xenograft）和PDX（Patient-derived xenograft）模型中进行的治疗效果评估显示，miR-449甲基化失活组对于表观药物呈现良好的应答。综上所述，本项目揭示了表观药物抑制肝癌细胞生长的分子机制，同时发现miR-449启动子区域甲基化修饰与表达水平可作为表观药物使用的标准。此外，在本项经费支持下，申请者对核仁蛋白NOP14在胰腺癌中的功能进行研究，结果发现NOP14作为RNA结合蛋白调控TP53 mRNA稳定性，进而介导miR-17-P21通路活性。该研究提示，NOP14可作为携带TP53突变胰腺癌的潜在治疗靶标。本项目初始设想旨在探讨miR-5p/3p加工过程中的“Arm-switch”，因此研究系统分析了胃癌细胞中的miR-5p/3p的表达特征，结果显示，miR-5p/3p共表达的miRNA仅占很低比例，经典的加工成熟机制仍占主导地位。PVT1是本研究关注的另一个lncRNA，其加工为成熟miRNA过程与吉西他滨耐药相关。