外泌体传递circBIRC6调控GRIN2D影响自噬介导胃癌恶性表型的机制研究

The exosomes secreted by cancer cells play an important role in the development and progression of cancer. Our previous studies verified that circBIRC6 was not only highly expressed in gastric cancer tissue but also enriched in the exosomes secreted by gastric cancer cells. It delivered by the exosomes to receptor cells for competitive binding with miR-488 to facilitate GRIN2D expression, promoting the migration and invasion of gastric cancer by blocking autophagy. On the basic of these contents, we will investigate the regulatory mechanism of circBIRC6-miR-488-GRIN2D axis on autophagy factor by bioinformatics, RNA co-precipitation, two-factor phenotype rescue experiment and two-factor mechanism rescue experiment, and explore the influence of the interaction between GRIN2D and MYO1E for downstream molecules by combining proteomic profiling, co-immunoprecipitation and fluorescent-multiplex-immunohistochemistry with clinical samples. Further experiments will elucidate the regulatory mechanism of exosome-transmitted circBIRC6 on the development of gastric cancer by affecting GRIN2D, and demonstrate that circBIRC6 and GRIN2D may represent novel target for precise diagnosis and treatment of gastric cancer. Finally, it can provide new ideas for the therapy of gastric cancer.

癌细胞分泌的外泌体在肿瘤发生发展中起重要作用。我们前期研究发现circBIRC6不仅在胃癌组织中高表达，而且在胃癌细胞分泌的外泌体中富集，并可由外泌体携带circBIRC6传递至受体细胞与miR-488竞争性结合解除miR-488对GRIN2D的抑制作用，从而使自噬受阻促进胃癌的侵袭迁移。课题拟在此基础上，结合生物信息学、RNA免疫共沉淀、双因素表型挽救实验和双因素机制挽救实验等方法验证circBIRC6-miR-488-GRIN2D轴对自噬因子的调控机制，并通过蛋白质谱、免疫共沉淀、多光谱免疫病理结合临床样本探究GRIN2D与MYO1E相互作用对下游分子的影响，从而阐明外泌体传递circBIRC6调控GRIN2D轴对胃癌发展的调控机制，论证circBIRC6及GRIN2D作为胃癌诊疗新靶点的可能性，为胃癌治疗提供新思路。

环状RNA（circRNA）是一类新型的非编码RNA，在肿瘤发生和进展中具有重要作用。我们前期研究发现circBIRC6不仅在胃癌组织中高表达，而且在胃癌细胞circBIRC6与miR-488竞争性结合解除miR-488对GRIN2D的抑制作用，从而使自噬受阻促进胃癌的侵袭迁移，但其具体调控机制尚不清楚。本研究，应用circRAN的鉴定及定量检测、RNA荧光原位杂交、RNAscope分析、PCR、RNA免疫沉淀、双荧光素酶报告基因检测、多重免疫荧光等技术展开研究，发现circBIRC6过表达增加GRIN2D表达，促进CAV1的表达，从而由于miR-488吸收导致自噬缺陷；这反过来又影响体内的肿瘤发生。总体而言，我们的研究结果表明，CircBIRC6-miR-488-GRIN2D轴促进胃癌细胞中CAV1的表达，导致自噬减少。故，circBIRC6和GRIN2D是胃癌患者的潜在治疗靶点和独立预后因素，此项目对胃癌的防治提供靶点和实验依据。