Table_5_Mdfi Promotes C2C12 Cell Differentiation and Positively Modulates Fast-to-Slow-Twitch Muscle Fiber Transformation.XLSX

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。肌分化因子家族抑制剂（Mdfi）可在NIH3T3细胞中抑制肌源性调节因子，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）C2C12细胞系，并对Mdfi-OE与野生型（WT）C2C12细胞开展了RNA测序（RNA-seq）。RNA测序结果显示，钙信号通路为最显著富集的信号通路。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（hub genes）。进一步的RNA测序与实验验证均证实，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。本研究还发现，Mdfi对快肌纤维向慢肌纤维转化的正向调控作用，是通过Myod、钙/钙调蛋白依赖性蛋白激酶2β（Camk2b）及其下游基因介导的，相关下游基因包括过氧化物酶体增殖物激活受体γ共激活因子1α（Pgc1a）、丙酮酸脱氢酶激酶4（Pdk4）、柠檬酸合酶（Cs）、细胞色素c氧化酶亚基4（Cox4）、酰基辅酶A脱氢酶（Acadm）、酰基辅酶A氧化酶1（Acox1）、细胞色素c（Cycs）及ATP合酶亚基α1（Atp5a1）。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转化。本研究结果进一步阐明了Mdfi在肌源性发育及肌纤维类型转化中的调控机制，可为肌肉相关及代谢相关疾病提供潜在的治疗靶点。