SNAP-23的O-GlcNAc修饰介导肝母细胞瘤释放外泌体的机制及其临床诊断价值研究

Hepatoblastoma (HB) is a common hepatic malignant tumor in childhood, of which the diagnostic and therapeutic methods are unitary, especially lacking valuable molecular markers, due to the fact that its pathogenesis is unclear. In recent years, many studies of the declarer team have shown that O-GlcNAcylation, a method of monosaccharide modification of proteins, is closely related to the energy metabolism of tumor cells. Our previous studies found that, compared with normal liver tissues, HB tissues had a higher level of O-GlcNAcylation, besides, promoting HB cells to release more exosomes through the O-GlcNAcylation of a pivotal protein SNAP-23 which was generated and released by exosomes. This project intends to deeply elucidate the relation of O-GlcNAcylation and HB through in vivo and in vitro assays, and probe the possibility of serum exosomal circRNAs (exo-circRNAs) of HB patients to serve as a molecular marker of HB prognosis by identifying the circular RNAs (circRNAs) in exosomes via a high-throughput sequencing method, therefore providing a novel approach for HB diagnosis.

肝母细胞瘤(hepatoblastoma, HB)是儿童期常见的肝脏恶性肿瘤，由于其发病机制不清楚，因此诊疗手段单一，尤其缺乏有价值的分子标志物。O-GlcNAc糖基化（O-GlcNAcylation）是针对蛋白的一种单糖基修饰方式，近年申报人团队的多项研究发现其与肿瘤细胞的能量代谢密切相关。本课题组前期研究发现HB较正常组织存在较高水平的O-GlcNAc修饰，并能通过调控外泌体释放的关键蛋白SNAP-23的O-GlcNAc修饰促进HB细胞释放更多外泌体，并从外泌体中检测到环状RNAs(circular RNAs，circRNAs)。本项目拟通过体内外方法深入解析O-GlcNAc修饰与HB的关系，通过高通量测序鉴定外泌体中的环状RNAs种类, 探讨患儿血清外泌体circRNAs(exo-circRNAs)作为HB诊断与预后的分子标志物的可能性，为肝母细胞瘤的临床诊断提供新方法。

本项目拟通过体内外方法深入解析O-GlcNAc修饰与HB的关系，通过高通量测序鉴定外泌体中的环状RNAs种类，探讨患儿血清外泌体circRNAs(exo-circRNAs)作为HB诊断与预后的分子标志物的可能性，为肝母细胞瘤的临床诊断提供新方法。经实验验证，我们初步明确，HB组织中O-GlcNAc修饰水平显著增强并促进肿瘤增殖、转移和侵袭。HB细胞中外泌体释放显著增加，并且该外泌体增加由O-GlcNAc修饰蛋白SNAP-23介导。O-GlcNAc修饰调控SNAP-23的蛋白稳定性，促进其表达，维持HB细胞分泌外泌体的功能。加入HB细胞分泌的外泌体可以显著挽救SNAP-23蛋白敲除后的生长抑制效应。同时，高通量测序鉴定出一系列患儿血清外泌体circRNAs，或可作为HB诊断与预后的分子标志物，为肝母细胞瘤的临床诊断提供新方法。