Table_1_Mdfi Promotes C2C12 Cell Differentiation and Positively Modulates Fast-to-Slow-Twitch Muscle Fiber Transformation.DOCX

Muscle development requires myoblast differentiation and muscle fiber formation. Myod family inhibitor (Mdfi) inhibits myogenic regulatory factors in NIH3T3 cells, but how Mdfi regulates myoblast myogenic development is still unclear. In the present study, we constructed an Mdfi-overexpression (Mdfi-OE) C2C12 cell line by the CRISPR/Cas9 system and performed RNA-seq on Mdfi-OE and wild-type (WT) C2C12 cells. The RNA-seq results showed that the calcium signaling pathway was the most significant. We also established the regulatory networks of Mdfi-OE on C2C12 cell differentiation and muscle fiber type transformation and identified hub genes. Further, both RNA-seq and experimental verification demonstrated that Mdfi promoted C2C12 cell differentiation by upregulating the expression of Myod, Myog, and Myosin. We also found that the positive regulation of Mdfi on fast-to-slow-twitch muscle fiber transformation is mediated by Myod, Camk2b, and its downstream genes, such as Pgc1a, Pdk4, Cs, Cox4, Acadm, Acox1, Cycs, and Atp5a1. In conclusion, our results demonstrated that Mdfi promotes C2C12 cell differentiation and positively modulates fast-to-slow-twitch muscle fiber transformation. These findings further our understanding of the regulatory mechanisms of Mdfi in myogenic development and muscle fiber type transformation. Our results suggest potential therapeutic targets for muscle- and metabolic-related diseases.

肌肉发育依赖于成肌细胞分化与肌纤维形成。肌分化因子（Myod）家族抑制剂（Mdfi）可在NIH3T3细胞中抑制肌源性调控因子，但Mdfi如何调控成肌细胞的肌源性发育，目前仍不明确。本研究通过CRISPR/Cas9系统构建了Mdfi过表达（Mdfi-OE）的C2C12细胞系，并对Mdfi-OE与野生型（WT）C2C12细胞开展RNA测序（RNA-seq）。RNA-seq结果显示，钙信号通路为富集程度最高的通路。本研究还构建了Mdfi-OE调控C2C12细胞分化与肌纤维类型转化的调控网络，并鉴定得到核心枢纽基因。进一步的RNA-seq分析与实验验证均证实，Mdfi可通过上调肌分化因子（Myod）、肌细胞生成素（Myog）及肌球蛋白（Myosin）的表达，促进C2C12细胞分化。本研究同时发现，Mdfi对快肌纤维向慢肌纤维转化的正向调控作用，经由Myod、钙/钙调蛋白依赖性蛋白激酶2β（Camk2b）及其下游基因（如Pgc1a、Pdk4、Cs、Cox4、Acadm、Acox1、Cycs及Atp5a1）介导。综上，本研究结果证实，Mdfi可促进C2C12细胞分化，并正向调控快肌纤维向慢肌纤维的类型转化。上述发现进一步深化了我们对Mdfi在肌源性发育及肌纤维类型转化中调控机制的认知，同时为肌肉相关与代谢相关疾病提供了潜在治疗靶点。