Central Laboratory, Peking University School and Hospital of Stomatology, Beijing, China.
National Engineering Laboratory for Digital and Material Technology of Stomatology, Peking University School and Hospital of Stomatology, Beijing, China.
PeerJ. 2022 May 3;10:e13360. doi: 10.7717/peerj.13360. eCollection 2022.
MicroRNAs play an important role in myogenic differentiation, they bind to target genes and regulate muscle formation. We previously found that miR-9-5p, which is related to bone formation, was increased over time during the process of myogenic differentiation. However, the mechanism by which miR-9-5p regulates myogenic differentiation remains largely unknown. In the present study, we first examined myotube formation and miR-9-5p, myogenesis-related genes including Dlx3, Myod1, Mef2c, Desmin, MyoG and Myf5 expression under myogenic induction. Then, we detected the expression of myogenic transcription factors after overexpression or knockdown of miR-9-5p or Dlx3 in the mouse premyoblast cell line C2C12 by qPCR, western blot and myotube formation under myogenic induction. A luciferase assay was performed to confirm the regulatory relationships between not only miR-9-5p and Dlx3 but also Dlx3 and its downstream gene, Myf5, which is an essential transcription factor of myogenic differentiation. The results showed that miR-9-5p promoted myogenic differentiation by increasing myogenic transcription factor expression and promoting myotube formation, but Dlx3 exerted the opposite effect. Moreover, the luciferase assay showed that miR-9-5p bound to the 3'UTR of Dlx3 and downregulated expression. Dlx3 in turn suppressed Myf5 expression by binding to the Myf5 promoter, ultimately inhibiting the process of myogenic differentiation. In conclusion, the miR-9-5p/Dlx3/Myf5 axis is a novel pathway for the regulation of myogenic differentiation, and can be a potential target to treat the diseases related to muscle dysfunction.
微小 RNA 在肌生成分化中发挥重要作用，它们与靶基因结合并调节肌肉形成。我们之前发现，与骨形成有关的 miR-9-5p 在肌生成分化过程中随着时间的推移而增加。然而，miR-9-5p 调节肌生成分化的机制在很大程度上仍然未知。在本研究中，我们首先检查了肌管形成和 miR-9-5p、肌生成相关基因（包括 Dlx3、Myod1、Mef2c、Desmin、MyoG 和 Myf5）在肌生成诱导下的表达。然后，我们通过 qPCR、western blot 和肌生成诱导下的肌管形成检测了 miR-9-5p 或 Dlx3 过表达或敲低后小鼠前成肌细胞系 C2C12 中的肌生成转录因子的表达。通过荧光素酶测定进一步证实了不仅 miR-9-5p 和 Dlx3 之间，而且 Dlx3 和其下游基因 Myf5 之间的调节关系，Myf5 是肌生成分化的关键转录因子。结果表明，miR-9-5p 通过增加肌生成转录因子的表达和促进肌管形成来促进肌生成分化，但 Dlx3 则产生相反的效果。此外，荧光素酶测定表明，miR-9-5p 与 Dlx3 的 3'UTR 结合并下调其表达。Dlx3 反过来通过结合 Myf5 启动子抑制 Myf5 表达，最终抑制肌生成分化过程。总之，miR-9-5p/Dlx3/Myf5 轴是调节肌生成分化的新途径，可能成为治疗与肌肉功能障碍相关疾病的潜在靶点。
