环状RNA调控RBM8A活性抑制肝素酶蛋白翻译对胃癌侵袭转移的作用及机制研究

Previous studies reveal that the N6-methyladenosine (m6A) modification within 5'-untranslated region facilitates protein translation, while circular RNA (circRNA) is able to regulate the expression of parental gene through interacting with RNA binding protein. Our results indicated that in gastric cancer, circRNA derived from exons of heparanase (circ-HPSE) was able to interact with RNA binding motif protein 8A (RBM8A) and block its recruitment of RNA methyltransferase like 3 (METTL3) on the 5'-untranslated region of HPSE, resulting in suppression of m6A modification and HPSE protein translation. Therefore, this study will further elucidate the effects of RBM8A and METTL3 on the m6A modification within 5'-untranslated region and protein translation of HPSE in gastric cancer. The domains essential for the interaction of RBM8A with circ-HPSE and METTL3 will be explored. The impacts of circ-HPSE on RBM8A-recruited METTL3 enrichment on regulatory sites, m6A modification, and HPSE expression will be studied in gastric cancer. Through ectopic expression or knockdown of circ-HPSE and rescue of HPSE levels or RBM8A function, the changes in the invasion, metastasis, and angiogenesis of gastric cancer cells will be detected in vitro and in vivo. This study will define the roles and mechanisms of circRNA in regulating the expression of HPSE, and will provide a novel strategy for the treatment of gastric cancer.

研究显示：5'-非翻译区N6-甲基腺嘌呤(m6A)修饰可促进蛋白翻译；而环状RNA(circRNA)能通过与RNA结合蛋白相互作用，调控亲本基因表达。我们发现：胃癌中肝素酶(HPSE)外显子生成的circ-HPSE能结合RNA结合基序蛋白8A(RBM8A)，阻遏RBM8A招募RNA甲基化酶3(METTL3)至HPSE的5'-非翻译区，抑制m6A修饰及HPSE蛋白翻译。据此，本研究拟深入解析RBM8A、METTL3对胃癌中HPSE的5'-非翻译区m6A修饰及蛋白翻译的调控作用；明确RBM8A与circ-HPSE、METTL3相互作用的结构域；观测circ-HPSE对RBM8A招募METTL3至调控位点、m6A修饰及HPSE表达的影响；观测过表达/敲低circ-HPSE、回补HPSE表达或RBM8A功能对胃癌侵袭转移的调控作用，从环状RNA角度阐明HPSE表达调控机理，为胃癌的治疗提供新思路。

胃癌是我国常见的消化道恶性肿瘤之一，每年约40万新发病例，占全球总发病例数的42%，严重危害人类身体健康。解析胃癌发生、发展的分子机制及防治策略已成为国内外研究的焦点。乙酰肝素酶(HPSE)是目前发现的唯一降解硫酸乙酰肝素蛋白多糖的β-D-葡萄糖醛酸内切酶，能导致细胞外基质降解和重塑，释放生物活性因子，促进肿瘤侵袭转移，但HPSE蛋白翻译的调控机制不明。本研究发现：在胃癌组织和细胞中，RNA结合基序蛋白8A(RBM8A)、RNA甲基化酶3(METTL3)表达上调；免疫共沉淀、RNA免疫沉淀、RNA甲基化免疫沉淀检测显示：RBM8A与METTL3存在蛋白相互作用，能招募METTL3至HPSE的5'-非翻译区(5'-UTR)，促进m6A修饰及HPSE蛋白翻译；而HPSE外显子生成的circ-HPSE在胃癌组织和细胞中低表达，能结合RBM8A，阻遏RBM8A、METTL3在HPSE 5'-UTR的募集，降低HPSE蛋白水平；通过建立稳定转染circ-HPSE过表达载体的胃癌细胞株模型，发现过表达circ-HPSE能抑制胃癌细胞的体内外增殖、侵袭及转移活性，阻遏胃癌恶性进展。因此，本项目揭示了circ-HPSE能够通过抑制RBM8A活性、m6A修饰对胃癌中HPSE表达的调控作用及机制，丰富了HPSE蛋白翻译调控机理，为胃癌的治疗提供了新靶点和新途径，具有潜在的应用前景和社会效益。