YTHDF2介导hsa_circ_0015508去稳定化增强miR-496对FOXN3的抑制从而驱动鼻咽癌进展

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中文摘要

目的： YTH N6-甲基腺苷RNA结合蛋白F2（YTHDF2）已被认为与鼻咽癌（NPC）进展相关。越来越多的证据表明，许多环状RNA（circRNA）参与调控肿瘤进展。然而，YTHDF2对circRNA的调控如何促进NPC进展仍有待揭示。本研究旨在阐明YTHDF2介导的circRNA调控在NPC迁移和侵袭中的作用及机制。方法：使用GEO数据集和免疫组化评估NPC中YTHDF2的表达。在HNE1和5-8F细胞中进行功能实验，通过伤口愈合和transwell实验评估迁移/侵袭，通过Western blotting检测上皮-间质转化（EMT）标志物。通过circRNA测序筛选候选circRNA。通过甲基化RNA免疫沉淀-定量PCR（MeRIP-qPCR）评估circRNA m6A修饰。使用SELECT方法（基于单碱基延伸和连接的qPCR扩增）鉴定特异性m6A修饰位点。通过放线菌素D实验测量RNA稳定性。通过荧光素酶报告基因实验验证circRNA-miRNA和miRNA-靶标相互作用。结果：YTHDF2在NPC组织中表达上调，并发现可促进体外迁移和侵袭表型。机制上，m6A修饰的circ_0015508被YTHDF2识别并降解。YTHDF2介导的促迁移和促侵袭效应被circ_0015508逆转。FOXN3被鉴定为miR-496的靶标。发现circ_0015508可吸附miR-496，从而解除对FOXN3表达的抑制。结论：本研究描绘了一条潜在的致癌通路，其中YTHDF2通过降解circ_0015508促进NPC迁移和侵袭，从而消除miR-496的海绵效应并抑制FOXN3表达。

英文摘要

Objectives: YTH N6-Methyladenosine RNA Binding Protein F2 (YTHDF2) had been implicated in nasopharyngeal carcinoma (NPC) progression. Increasing evidence indicated that numerous circular RNAs (circRNAs) were involved in regulating tumor progression. However, how the regulation of circRNAs by YTHDF2 contributes to NPC progression remains to be uncovered. In this study, we aimed to elucidate the role and mechanism of YTHDF2-mediated circRNA regulation in NPC migration and invasion. Methods: YTHDF2 expression in NPC was assessed using GEO datasets and immunohistochemistry. Functional experiments were performed in HNE1 and 5-8F cells, with migration/invasion evaluated by wound healing and transwell assays, and epithelial-mesenchymal transition (EMT) markers by Western blotting. CircRNA candidates were screened through circRNA sequencing. CircRNA m6A modification was assessed by methylated RNA immunoprecipitation-quantitative PCR (MeRIP-qPCR). The SELECT method (single-base elongation- and ligation-based qPCR amplification) was used to identify the specific m6A modification site. RNA stability was measured by actinomycin D assay. circRNA-miRNA and miRNA-target interactions were validated by luciferase reporter assays. Results: YTHDF2 expression was upregulated in NPC tissues and was found to promote in vitro migration and invasion phenotypes. Mechanistically, m6A-modified circ_0015508 was recognized and degraded by YTHDF2. YTHDF2-mediated pro-migration and pro-invasion effects were reversed by circ_0015508. FOXN3 was identified as a target of miR-496. Circ_0015508 was found to sequester miR-496, thereby de-repressing FOXN3 expression. Conclusion: In this study, a potential oncogenic pathway was delineated in which NPC migration and invasion were facilitated by YTHDF2 via degradation of circ_0015508, thereby the sponging effect of miR-496 was abolished and FOXN3 expression was suppressed.