LncRNA AFAP1-AS1通过ceRNA活性参与食道腺癌细胞凋亡和自噬的机制研究

Long noncoding RNAs (lncRNAs) have been the research hotspot in recent years. However, its role in esophageal adenocarcinoma (EAC) is unclear. The competing endogenous RNA (ceRNA) hypothesis indicates that lncRNAs may act as endogenous sponges for miRNAs to inhibit activaty of miRNAs, and affect the distribution of miRNAs on their targets in further, thereby being involved in tumor development and progression. In previous study, using the next generation sequencing technology we identified a lncRNA，AFAP1-AS1, which is highly expressed in Barrett’s esophagus(BE) and EAC. Inhibition of the expression of AFAP1-AS1 can cause the attenuation of proliferation, colony formation, migration and invasion in EAC cells. Notably, downregulation of AFAP1-AS1 can induce significant cell apoptosis by activating Caspase-3. However, the molecular mechanism how AFAP1-AS1 regulates cell apoptosis progress has not been clarified. Bioinformatics analysis results show that there are some miRNAs binding sites including miR-429, suggesting possible interaction between lncRNA AFAP1-AS1 and miR-429. Therefore, we hypothesize that lncRNA AFAP1-AS1 may function as a ceRNA to compete with miR-429 activity, and then regulate the expression of the miR-429 target gene Bcl-2, which is one of the key regulators of cell apoptosis and autophagy. In this study, we will stably knock down the AFAP1-AS1 expression in EAC cell lines and investigate whether lncRNA AFAP1-AS1 regulate cell apoptosis and autophagy by acting as a ceRNA for miRNA. In sum, our study will provide an insight on revealing the underlying mechanism how lncRNAs regulate cell apoptosis and autophagy, and discovering new target in EAC therapy.

长链非编码RNA（lncRNA）是一类参与肿瘤发生发展的重要调节分子。文献报道lncRNA可作为ceRNA通过miRNA调控靶基因表达，进而影响肿瘤进程。申请者前期研究发现，lncRNA AFAP1-AS1表达下调可以抑制食道腺癌细胞增殖、克隆形成、侵袭迁移，促进细胞凋亡，但其分子机制尚未完全阐明。生物信息学分析发现AFAP1-AS1序列含有miR-429的种子序列，提示在食道腺癌细胞中AFAP1-AS1可能通过发挥ceRNA活性，竞争性结合miR-429，调控miR-429的靶基因Bcl-2的表达，影响食道腺癌细胞的凋亡和自噬过程。本项目拟通过建立稳定抑制AFAP1-AS1表达的食道腺癌细胞株，采用流式细胞术、荧光素酶报告基因活性检测、RNA免疫共沉淀等方法探讨AFAP1-AS1是否作为ceRNA发挥对细胞凋亡和自噬的调控，从而为食道腺癌的治疗提供新思路。