Department of Stomatology, The First Hospital of Putian City, 449 Nanmen West Road, Chengxiang District, Putian City, Putian, 351100, China.
Department of Neurology, The Affiliated Hospital of Putian University, Putian, 351100, China.
BMC Oral Health. 2024 May 28;24(1):625. doi: 10.1186/s12903-024-04388-2.
BACKGROUND: Oral squamous cell cancer (OSCC) is a prevalent malignancy in oral cavity, accounting for nearly 90% of oral malignancies. It ranks sixth among the most common types of cancer worldwide and is responsible for approximately 145,000 deaths each year. It is widely accepted that noncoding RNAs participate cancer development in competitive regulatory interaction, knowing as competing endogenous RNA (ceRNA) network, whereby long non-coding RNA (lncRNA) function as decoys of microRNAs to regulate gene expression. LncRNA FOXD2-AS1 was reported to exert an oncogenic role in OSCC. Nevertheless, the ceRNA network mediated by FOXD2-AS1 was not investigated yet. This study aimed to explore the effect of FOXD2-AS1 on OSCC cell process and the underlying ceRNA mechanism. METHODS: FOXD2-AS1 expression in OSCC cells were determined via reverse transcription and quantitative polymerase chain reaction. Short hairpin RNA targeting FOXD2-AS1 was transfected into OSCC cells to silence FOXD2-AS1 expression. Then, loss-of-function experiments (n = 3 each assay) were performed to measure cell proliferation, apoptosis, migration, and invasion using colony formation, TdT-mediated dUTP Nick-End Labeling, wound healing and Transwell assays, respectively. RNA binding relation was verified by RNA immunoprecipitation and luciferase reporter assays. Rescue experiments were designed to validate whether FOXD2-AS1 affects cell behavior via the gene cellular retinoic acid binding protein 2 (CRABP2). Statistics were processed by GraphPad Prism 6.0 Software and SPSS software. RESULTS: FOXD2-AS1 was significantly upregulated in Cal27 and SCC9 cells (6.8 and 6.4 folds). In response to FOXD2-AS1 knockout, OSCC cell proliferation, migration and invasion were suppressed (approximately 50% decrease) while OSCC cell apoptosis was enhanced (more than two-fold increase). FOXD2-AS1 interacted with miR-378 g to alter CRABP2 expression. CRABP2 upregulation partly rescued (*p < 0.05, **p < 0.01, ***p < 0.001) the inhibitory impact of FOXD2-AS1 depletion on malignant characteristics of OSCC cells. CONCLUSION: FOXD2-AS1 enhances OSCC malignant cell behaviors by interacting with miR-378 g to regulate CRABP2 expression.
背景：口腔鳞状细胞癌（OSCC）是口腔中一种常见的恶性肿瘤，占口腔恶性肿瘤的近 90%。它在全球最常见的癌症类型中排名第六，每年导致约 14.5 万人死亡。人们普遍认为非编码 RNA 以竞争性调节相互作用的方式参与癌症的发生发展，即称为竞争内源性 RNA（ceRNA）网络，其中长非编码 RNA（lncRNA）作为 microRNA 的诱饵发挥作用，调节基因表达。已有报道称 lncRNA FOXD2-AS1 在 OSCC 中发挥致癌作用。然而，FOXD2-AS1 介导的 ceRNA 网络尚未被研究。本研究旨在探讨 FOXD2-AS1 对 OSCC 细胞过程的影响及其潜在的 ceRNA 机制。
方法：通过逆转录和定量聚合酶链反应检测 OSCC 细胞中的 FOXD2-AS1 表达。转染靶向 FOXD2-AS1 的短发夹 RNA 以沉默 FOXD2-AS1 表达。然后，通过集落形成、TdT 介导的 dUTP 末端标记、划痕愈合和 Transwell 分析分别进行失活功能实验（每组实验重复 3 次），以测量细胞增殖、凋亡、迁移和侵袭。通过 RNA 免疫沉淀和荧光素酶报告基因测定验证 RNA 结合关系。设计挽救实验以验证 FOXD2-AS1 是否通过基因细胞视黄酸结合蛋白 2（CRABP2）影响细胞行为。统计分析采用 GraphPad Prism 6.0 软件和 SPSS 软件进行。
结果：FOXD2-AS1 在 Cal27 和 SCC9 细胞中显著上调（6.8 和 6.4 倍）。FOXD2-AS1 敲除后，OSCC 细胞增殖、迁移和侵袭受到抑制（约 50%减少），而 OSCC 细胞凋亡增强（增加两倍以上）。FOXD2-AS1 与 miR-378g 相互作用改变了 CRABP2 的表达。CRABP2 的上调部分挽救了（*p<0.05，**p<0.01，***p<0.001）FOXD2-AS1 耗竭对 OSCC 细胞恶性特征的抑制作用。
结论：FOXD2-AS1 通过与 miR-378g 相互作用调节 CRABP2 表达，增强 OSCC 恶性细胞行为。
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