In eukaryotes, N6-methyladenosine (m⁶A) is one of the most abundant modifications on RNAs, and it plays important roles in many biological processes and diseases such as cancer. While most m⁶A researches focus on message RNAs and long non-coding RNAs, recent studies have reported the presence of m⁶A in small RNAs. Nevertheless, current knowledge about m⁶A prevalence in mature microRNAs (miRNA) is extremely limited and the functional significance of m⁶A methylation in miRNAs remains to be elucidated. Here, we demonstrated cell-specific m⁶A profiles of miRNAs in A549 human non-small cell lung cancer (NSCLC) cells and HEK293A cells by using miRNA m⁶A immunoprecipitation sequencing and constructed the consensus motif in m⁶A-enriched miRNAs de novo. We found that miR-21–5p, an oncogenic miRNA, showed the highest m⁶A enrichment in NSCLC cells. Depletion of the demethylase ALKBH5 did not change the expression level of miR-21–5p, but altered the m⁶A abundance of miR-21–5p, thereby changing the expression levels of its target gene. We further synthesized m⁶A modified miR-21–5p mimics in vitro and demonstrated that in NSCLC cells, m⁶A marks in mature miR-21–5p could directly affect its silencing potency towards target genes, which finally impaired its promotion to proliferation and motility. Together, our findings reveal the landscape of m⁶A modification in mature miRNAs, and provide the first evidence that it may contribute to the mRNA responses to cancer-related miRNAs.

在真核生物中，N6-甲基腺苷 (m ⁶ A) 是 RNA 上最丰富的修饰之一，它在许多生物过程和疾病（如癌症）中发挥着重要作用。虽然大多数 m ⁶ A 研究集中在信息 RNA 和长链非编码 RNA，但最近的研究报告了 m ⁶ A 在小 RNA 中的存在。然而，目前关于成熟 microRNA (miRNA) 中m ⁶ A 流行的知识非常有限，并且 miRNA 中 m ⁶ A 甲基化的功能意义仍有待阐明。在这里，我们通过使用 miRNA m ⁶证明了 A549 人非小细胞肺癌 (NSCLC) 细胞和 HEK293A 细胞中 miRNA 的细胞特异性 m ⁶ A 谱免疫沉淀测序并从头构建富含m ⁶ A 的 miRNA的共有基序。我们发现 miR-21-5p 是一种致癌 miRNA，在 NSCLC 细胞中显示出最高的 m ⁶ A 富集。去甲基化酶ALKBH5的消耗没有改变miR-21-5p的表达水平，但改变了miR-21-5p的m ⁶ A丰度，从而改变了其靶基因的表达水平。我们进一步在体外合成了 m ⁶ A 修饰的 miR-21-5p 模拟物，并证明在 NSCLC 细胞中，m ⁶成熟 miR-21-5p 中的 A 标记可直接影响其对靶基因的沉默效力，最终削弱其对增殖和运动的促进作用。总之，我们的发现揭示了成熟 miRNA中 m ⁶ A 修饰的景观，并提供了第一个证据，表明它可能有助于对癌症相关 miRNA 的 mRNA 反应。