Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
Int J Biol Macromol. 2019 Jan;121:127-134. doi: 10.1016/j.ijbiomac.2018.10.017. Epub 2018 Oct 3.
Using bioinformatics analysis, we found some mature human miRNAs containing G-rich sequences with four G-tracts that had a high probability of forming G-quadruplex structures. Here, we chose G-rich miR-1587 as a model to characterize the function and regulation of miRNAs. Using electrospray ionization mass spectrometry, magnetic resonance imaging, circular dichroism spectrometry, we had confirmed that miR-1587 folded into a stable parallel G-quadruplex structure. By microarray, Q-RT-PCR and 3'UTR luciferase assay, TAGLN, an early marker of smooth muscle differentiation and tumor suppressor, was identified as a target gene of miR-1587, thus providing a direct target to study miR-1587 functions. We identified three aspects of miR-1587 regulation: 1) KCl induced miR-1587 G-quadruplex formation, reducing the interaction between miR-1587 and the target gene, and inhibiting miR-1587 function; 2) pseudopalmatine ligand further inhibited miR-1587 binding to TAGLN mRNA, which disrupted its function and increased the TAGLN expression; 3) the addition of TMPyP4 ligand interfered G-quadruplex formation, and significantly enhanced miR-1587 regulation of TAGLN expression. This study has revealed the possibility of using the G-quadruplex structure as a strategy to regulate miR-1587 function, showing potential for the development of up- and downregulation of mature G-rich microRNA function by modulating its G-quadruplex and using small molecules.
利用生物信息学分析，我们发现了一些含有富含 G 序列的成熟人类 miRNA，其中四个 G 链段具有形成 G-四链体结构的高概率。在这里，我们选择富含 G 的 miR-1587 作为模型，以表征 miRNA 的功能和调控。通过电喷雾电离质谱、磁共振成像、圆二色光谱，我们已经证实 miR-1587 折叠成一种稳定的平行 G-四链体结构。通过微阵列、Q-RT-PCR 和 3'UTR 荧光素酶测定，我们鉴定出 TAGLN（平滑肌分化的早期标志物和肿瘤抑制因子）是 miR-1587 的靶基因，从而为研究 miR-1587 的功能提供了直接的靶基因。我们确定了 miR-1587 调控的三个方面：1）KCl 诱导 miR-1587 G-四链体形成，减少 miR-1587 与靶基因的相互作用，抑制 miR-1587 功能；2）假马齿苋碱配体进一步抑制 miR-1587 与 TAGLN mRNA 的结合，破坏其功能并增加 TAGLN 表达；3）TMPyP4 配体的加入干扰 G-四链体形成，显著增强 miR-1587 对 TAGLN 表达的调控。这项研究揭示了利用 G-四链体结构作为调控 miR-1587 功能的策略的可能性，为通过调节其 G-四链体和使用小分子来调控成熟 G-丰富 miRNA 功能的上调和下调提供了潜在的可能性。
