Long non-coding RNAs (lncRNAs) are key regulators of the 6-methyladenosine (m6A) epigenetic modification, playing a role in the initiation and progression of tumors. However, the regulatory mechanisms in head and neck squamous cell carcinoma (HNSCC) remain elusive. In this study, we investigated the molecular regulatory mechanisms of the lncRNA RASAL2-AS1 in the occurrence and development of HNSCC tumors. A bioinformatics analysis was conducted to analyze the expression level of RASAL2-AS1 in HNSCC and normal tissues. RASAL2-AS1 mRNA and protein levels were detected using RT-PCR and Western blotting. Wound healing, transwell assays, flow cytometry, M6A dot blot, and RNA immunoprecipitation experiments were conducted to explore the regulatory role of the RASAL2-AS1 and downstream targets METTL14/LIS1 signaling pathway in HNSCC. Immunohistochemical examination was conducted to evaluate the expression of METTL14 and LIS1 in HNSCC and normal tissues. A tumor xenograft model of BALB/c nude mice was established to assess the impact of RASAL2-AS1 on cell proliferation and growth. RASAL2-AS1 high expression in HNSCC and cells deteriorated with survival rates of HNSCC. RASAL2-AS1 overexpression in HNSCC accelerated cell migration, colony formation, cell proliferation, cell cycle in S stage, while RASAL2-AS1 knockdown in HNSC cells inhibited cell cycle in G1 stage. After silencing METTL14, the above effects induced by overexpression of the RASAL2-AS1 were reversed. RASAL2-AS1 overexpression prompted LIS1 expression, whereas RASAL2-AS1 silencing reduced LIS1 levels in HNSCC cells, which was confirmed by immunohistological staining. Results demonstrated elevated expression of METTL14 or LIS1 in tongue cancer tissues. Overexpression of RASAL2-AS1 promoted tumor weight and tumor volume, which was counteracted by pcDNA3.1 RASAL2-AS1 plus silencing METTL14 and METTL14 and LIS1 were significantly decreased. Our study highlights the functional importance of the LncRNA RASAL2-AS1 in HNSCC and might assist in the development of a prognostic stratification and therapeutic approach. Which regulates HNSCC with the dependence of m6a manner.

中文翻译：

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LncRNA RASAL2-AS1促进METTL14介导的m6A甲基化在头颈鳞状细胞癌增殖和进展中的作用

长非编码 RNA (lncRNA) 是 6-甲基腺苷 (m6A) 表观遗传修饰的关键调节因子，在肿瘤的发生和进展中发挥作用。然而，头颈鳞状细胞癌（HNSCC）的调节机制仍然难以捉摸。在本研究中，我们探讨了lncRNA RASAL2-AS1在HNSCC肿瘤发生发展中的分子调控机制。通过生物信息学分析RASAL2-AS1在HNSCC和正常组织中的表达水平。使用 RT-PCR 和蛋白质印迹法检测 RASAL2-AS1 mRNA 和蛋白质水平。通过伤口愈合、transwell实验、流式细胞术、M6A斑点印迹和RNA免疫沉淀实验来探讨RASAL2-AS1和下游靶标METTL14/LIS1信号通路在HNSCC中的调节作用。免疫组化检测HNSCC和正常组织中METTL14和LIS1的表达。建立BALB/c裸鼠肿瘤异种移植模型以评估RASAL2-AS1对细胞增殖和生长的影响。 RASAL2-AS1 在 HNSCC 中高表达，并且细胞随着 HNSCC 的存活率而恶化。 HNSCC中RASAL2-AS1的过表达加速了细胞迁移、集落形成、细胞增殖、S期细胞周期，而HNSC细胞中RASAL2-AS1敲除抑制了G1期细胞周期。沉默 METTL14 后，由 RASAL2-AS1 过表达引起的上述效应被逆转。 RASAL2-AS1 过表达促进了 LIS1 表达，而 RASAL2-AS1 沉默降低了 HNSCC 细胞中的 LIS1 水平，这一点通过免疫组织学染色得到证实。结果表明，舌癌组织中 METTL14 或 LIS1 的表达升高。 RASAL2-AS1 的过度表达会增加肿瘤重量和体积，而 pcDNA3.1 RASAL2-AS1 加沉默 METTL14 可以抵消这种影响，并且 METTL14 和 LIS1 显着降低。我们的研究强调了 LncRNA RASAL2-AS1 在 HNSCC 中的功能重要性，并可能有助于制定预后分层和治疗方法。其以m6a依赖性方式调节HNSCC。