Overexpression of Bmal1 promotes proliferation and differentiation of C2C12 myoblasts by enhancing the Wnt signaling

doi:10.13418/j.issn.1001-165x.2025.3.07

PDF(6890 KB)

Chinese Journal of Clinical Anatomy ›› 2025, Vol. 43 ›› Issue (3) : 278-288. DOI: 10.13418/j.issn.1001-165x.2025.3.07

Overexpression of Bmal1 promotes proliferation and differentiation of C2C12 myoblasts by enhancing the Wnt signaling

Jiang Xu¹, Ji Huihui¹, Song Yuting¹, Wang Liangliang², Li Na², Sun Junhong², Cui Huilin¹, Cao Ximei^1,3

Author information +

History +

Abstract

Objective To investigate the effect and mechanism of Bmal1 overexpression on C2C12 myoblasts proliferation and differentiation. Methods Bmal1 was overexpressed in C2C12 myoblasts by lentivirus-mediated stable transfection. The expression levels of mRNA and protein of some myogenic regulatory factors were determined by RT-qPCR or Western blot assay. Transcriptome sequence analysis and its related experiments were used to explore the expression of Wnt signaling pathway and cell cycle regulators in C2C12 myoblasts after overexpression of Bmal1 during the differentiation process. Subsequently, Wnt signaling pathway was blocked by inhibitor XAV939. The effects of XAV939 on C2C12 myoblasts after overexpression of Bmal1 were observed using corresponding experiments. Results Overexpression of Bmal1 promoted the proliferation of myoblasts cultured in growth medium. The S-phase fraction of total cells (SPF) and Proliferation Index (PI) of myoblasts increased. Compared with the blank control group and negative control group, the ratio of EdU positive cells and nuclear translocation of β-catenin were increased in C2C12 myoblasts after overexpression of Bmal1. Overexpression of Bmal1 significantly promoted myogenic differentiation and myotube fusion. The mRNA and protein expression levels of MyoD, myogenin, and Myh3 were up-regulated in a time-dependent manner in C2C12 myoblasts after overexpression of Bmal1. Cluster analysis and related experiments supported that the expression of the relative factors of Wnt signaling pathway and cell cycle was up-regulated during the proliferation and differentiation of Bmal1-overexpressed C2C12 myoblasts. XAV939(5μmol/L) treatment inhibited the Bmal1 overexpression-induced the up-regulated expression of Tcf1 and c-Jun. Continuous XAV939 treatment suppressed myotube fusion and down-regulated the expression of MyoD, myogenin, and Myh3 in Bmal1-overexpressed C2C12 myoblasts. Conclusions Bmal1 may serve as a potential target for skeletal muscle injury repair. Overexpression of Bmal1 enhances myoblasts proliferation and differentiation by enhancing Wnt signaling pathway.

Key words

Bmal1; / Wnt signaling pathway; / XAV939; / Proliferation; / Differentiation; / C2C12 myoblast

Cite this article

EndNote

Ris (Procite)

Bibtex

Download Citations

Jiang Xu, Ji Huihui, Song Yuting, Wang Liangliang, Li Na, Sun Junhong, Cui Huilin, Cao Ximei. Overexpression of Bmal1 promotes proliferation and differentiation of C2C12 myoblasts by enhancing the Wnt signaling[J]. Chinese Journal of Clinical Anatomy. 2025, 43(3): 278-288 https://doi.org/10.13418/j.issn.1001-165x.2025.3.07

References

[1] Yin L, Li N, Jia W, et al. Skeletal muscle atrophy: From mechanisms to treatments [J]. Pharmacol Res, 2021, 172: 105807. DOI: 10.1016/j.phrs.2021.105807.
[2] Careccia G, Mangiavini L, Cirillo F. Regulation of Satellite Cells Functions during Skeletal Muscle Regeneration: A Critical Step in Physiological and Pathological Conditions [J]. Int J Mol Sci, 2023, 25(1): 512. DOI: 10.3390/ijms25010512.
[3] Yoshimoto Y, Ikemoto-Uezumi M, Hitachi K, et al. Methods for Accurate Assessment of Myofiber Maturity During Skeletal Muscle Regeneration [J]. Front Cell Dev Biol, 2020, 8: 267. DOI: 10.3389/fcell.2020.00267.
[4] Schrader LA, Ronnekleiv-Kelly SM, Hogenesch JB, et al. Circadian disruption, clock genes, and metabolic health [J]. J Clin Invest, 2024, 134(14): e170998. DOI: 10.1172/JCI170998.
[5] Fernández-Martínez J, Ramírez-Casas Y, Aranda-Martínez P, et al. iMS-Bmal1-/- mice show evident signs of sarcopenia that are counteracted by exercise and melatonin therapies [J]. J Pineal Res, 2024, 76(1): e12912. DOI: 10.1111/jpi.12912.
[6] Gutierrez-Monreal MA, Wolff CA, Rijos EE, et al. Targeted Bmal1 restoration in muscle prolongs lifespan with systemic health effects in aging model [J]. JCI Insight, 2024, 9(22): e174007. DOI: 10.1172/jci.insight.174007.
[7] 傅泽铤, 夏雨, 丁海丽. 时钟基因BMAL1在运动性骨骼肌损伤恢复中的作用[J]. 中国应用生理学杂志, 2022, 38(3): 220-226. DOI: 10.12047/j.cjap.6243.2022.041.
Fu ZT, Xia Y, Ding HL. The role of clock gene BMAL1 in exercise-induced skeletal muscle injury recovery [J]. Chinese Journal of Applied Physiology, 2022, 38(3): 220-226. DOI: 10.12047/j.cjap.6243.2022.041.
[8] Zhou Q, Wang R, Su Y, et al. The molecular circadian rhythms regulating the cell cycle [J]. J Cell Biochem, 2024, 125(4): e30539. DOI: 10.1002/jcb.30539.
[9] Rabinovich-Nikitin I, Kirshenbaum LA. BMAL1 regulates cell cycle progression and angiogenesis of endothelial cells [J]. Cardiovasc Res, 2023, 119(10): 1889-1890. DOI: 10.1093/cvr/cvad103.
[10]叶翠, 吴海苗. BmaI1在经典Wnt通路调控间充质干细胞衰老中的作用[J]. 上海口腔医学, 2020,29(6): 601-605. DOI: 10.19439/j.sjos. 2020.06.008.
Ye C, Wu HM. The role of Bmal1 in regulating the aging of mesenchymal stem cells via Wnt/β-catenin signaling pathway [J]. Shanghai Journal of Stomatology, 2020, 29(6): 601-605. DOI: 10.19439/j.sjos.2020.06.008.
[11]Han Q, Li M, Su D, et al. Bmal1 regulates the stemness and tumorigenesis of gliomas with the Wnt/β-catenin signaling pathway [J]. Gene, 2025, 933: 148940. DOI: 10.1016/j.gene.2024.148940.
[12]Chatterjee S, Nam D, Guo B, et al. Brain and muscle Arnt-like 1 is a key regulator of myogenesis [J]. J Cell Sci. 2013, 126(Pt 10): 2213-2224. DOI: 10.1242/jcs.120519.
[13]杨新华, 闫银弟, 罗旭光, 等. 钟基因Bmal1、Clock参与调节骨骼肌的发育分化[J]. 中国组织工程研究, 2021, 25(20): 3130-3137. DOI: 10.3969/j.issn.2095-4344.3135.
Yang XH, Yan YD, Luo XG, et al. Bmal1 and Clock regulate the development and differentiation of skeletal muscle [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(20):3130-3137. DOI: 10.3969/j.issn.2095-4344.3135.
[14]Laothamatas I, Rasmussen ES, Green CB, et al. Metabolic and chemical architecture of the mammalian circadian clock [J]. Cell Chem Biol, 2023, 30(9): 1033-1052. DOI: 10.1016/j.chembiol.2023.08.014.
[15]Harfmann BD, Schroder EA, Esser KA. Circadian rhythms, the molecular clock, and skeletal muscle [J]. J Biol Rhythms, 2015, 30(2): 84-94. DOI: 10.1177/0748730414561638.
[16]Lin F, Chen Y, Li X, et al. Over-expression of circadian clock gene Bmal1 affects proliferation and the canonical Wnt pathway in NIH-3T3 cells [J]. Cell Biochem Funct, 2013, 31(2): 166-172. DOI: 10.1002/cbf.2871.
[17]Wherley TJ, Thomas S, Millay DP, et al. Molecular regulation of myocyte fusion [J]. Curr Top Dev Biol, 2024, 158: 53-82. DOI: 10.1016/bs.ctdb.2024.01.016.
[18]Tang J, Yang B, Song G, et al. Effect of bovine myosin heavy chain 3 on proliferation and differentiation of myoblast [J]. Anim Biotechnol, 2023, 34(9): 4337-4346. DOI: 10.1080/10495398.2022.2149549.
[19]Klement K, Brückner M, Bernkopf DB. Phosphorylation of axin within biomolecular condensates counteracts its tankyrase-mediated degradation [J]. J Cell Sci, 2023, 136(20): jcs261214. DOI: 10.1242/jcs.261214.
[20]Lin CS, Liang Y, Su SG, et al. Nucleoporin 93 mediates β-catenin nuclear import to promote hepatocellular carcinoma progression and metastasis [J]. Cancer Lett, 2022, 526: 236-247. DOI: 10.1016/j.canlet.2021.11.001.
[21]Yuan M, Shi H, Wang B, et al. Targeting SOCS2 alleviates myocardial fibrosis by reducing nuclear translocation of β-catenin [J]. Biochim Biophys Acta Mol Cell Res, 2024, 1871(7): 119804. DOI: 10.1016/j.bbamcr.2024.119804.
[22]Zhao XD, Shan Q, Xue HH. TCF1 in T cell immunity: a broadened frontier [J]. Nat Rev Immunol, 2022, 22(3): 147-157. DOI: 10.1038/s41577-021-00563-6.
[23]Nardi M, Baldelli S, Ciriolo MR, et al. Oleuropein Aglycone Peracetylated (3,4-DHPEA-EA(P)) Attenuates H(2)O(2)-Mediated Cytotoxicity in C2C12 Myocytes via Inactivation of p-JNK/p-c-Jun Signaling Pathway [J]. Molecules, 2020, 25(22): 5472. DOI: 10.1021/acs.jafc.1c04661.
[24]Hodge BA, Zhang X, Gutierrez-Monreal MA, et al. MYOD1 functions as a clock amplifier as well as a critical co-factor for downstream circadian gene expression in muscle [J]. Elife, 2019, 8: e43017. DOI: 10.7554/eLife.43017.
[25]Choi Y, Cho J, No MH, et al. Re-Setting the Circadian Clock Using Exercise against Sarcopenia [J]. Int J Mol Sci, 2020, 21(9): 3106. DOI: 10.3390/ijms21093106.