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MSTN Regulatory Network in Mongolian Horse Muscle Satellite Cells Revealed with miRNA Interference Technologies.

ABSTRACT: Myostatin (MSTN), a member of the transforming growth factor-β superfamily, inhibits the activation of muscle satellite cells. However, the role and regulatory network of MSTN in equine muscle cells are not well understood yet. We discovered that MSTN knockdown significantly reduces the proliferation rate of equine muscle satellite cells. In addition, after the RNA sequencing of equine satellite cells transfected with MSTN-interference plasmid and control plasmid, an analysis of the differentially expressed genes was carried out. It was revealed that MSTN regulatory networks mainly involve genes related to muscle function and cell-cycle regulation, and signaling pathways, such as Notch, MAPK, and WNT. Subsequent real-time PCR in equine satellite cells and immunohistochemistry on newborn and adult muscle also verified the MSTN regulatory network found in RNA sequencing analysis. The results of this study provide new insight into the regulatory mechanism of equine MSTN.

SUBMITTER: Budsuren U

PROVIDER: S-EPMC9601437 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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MSTN Regulatory Network in Mongolian Horse Muscle Satellite Cells Revealed with miRNA Interference Technologies.

Budsuren Undarmaa U Ulaangerel Tseweendolmaa T Shen Yingchao Y Liu Guiqin G Davshilt Toli T Yi Minna M Bold Demuul D Zhang Xinzhuang X Bai Dongyi D Dorjgotov Dulguun D Davaakhuu Gantulga G Jambal Tuyatsetseg T Li Bei B Du Ming M Dugarjav Manglai M Bou Gerelchimeg G

Genes 20221011 10

Myostatin (MSTN), a member of the transforming growth factor-β superfamily, inhibits the activation of muscle satellite cells. However, the role and regulatory network of MSTN in equine muscle cells are not well understood yet. We discovered that MSTN knockdown significantly reduces the proliferation rate of equine muscle satellite cells. In addition, after the RNA sequencing of equine satellite cells transfected with MSTN-interference plasmid and control plasmid, an ...[more]

PMID: 36292721

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Project description:Leucine has been shown to stimulate the mammalian/mechanistic target of rapamycin (mTOR) signaling pathway which plays numerous key regulatory roles in cell growth, survival, and metabolism including protein synthesis in a number of species. However, previous work with equine satellite cells has suggested distinct species differences in regards to physiological effects and the magnitude of responses to growth factors and regulators. Because there is limited research available regarding the role of leucine in regulating equine skeletal muscle protein synthesis, the objective of this study was to evaluate the effect of leucine on the mTOR signaling pathway in cultured equine satellite. Protein synthesis was evaluated by measuring the incorporation of [3H] Phenylalanine (3HPhe) in equine satellite cell myotube cultures treated with a leucine titration ranging from 0 to 408 µM. Our results show a 1.8-fold increase (P < 0.02) in protein synthesis at levels slightly greater than those found in the general circulation, 204 and 408 µM when compared to a no leucine control (0 µM). Puromycin incorporation, a nonradioactive surface sensing of translation (SUnSET) methodology, was also measured in cells treated with leucine (LEU; 408 µM), a no-leucine control (CON), and a puromycin-negative vehicle (PURO-). These results demonstrated a 180% increase (P = 0.0056) in puromycin incorporation in LEU compared to CON cultures. To evaluate the mTOR signaling pathway, equine satellite cell myotube cultures were treated with leucine (LEU; 408 µM) or a no-leucine control (CON) in the presence or absence of rapamycin (LR and CR, respectively), an inhibitor of mTOR. The mTOR inhibitor, rapamycin, suppressed phosphorylation of mTOR (P < 0.01) and rS6 (P < 0.01) with an increase in phosphorylation of rS6 in leucine-treated cultures observed when compared to control cultures (P < 0.05). Similarly, there was a 27% increase (P < 0.005) in the hyperphosphorylated γ-form of 4E-BP1 compared to total 4E-BP1 in LEU compared to CON cultures with leucine-induced phosphorylation of 4E-BP1 completely blocked by rapamycin with a smaller decrease observed in CR compared to CON cultures. The major finding of this study was that leucine activated the mTOR translation initiation pathway and increased transcription of global proteins in cultured equine satellite cells. Use of the cell culture system with primary equine muscle cell lines provides the opportunity to distinguish the impact of leucine on muscle and protein synthesis, independent of systemic interactions.

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Dataset Information

MSTN Regulatory Network in Mongolian Horse Muscle Satellite Cells Revealed with miRNA Interference Technologies.

Publications

MSTN Regulatory Network in Mongolian Horse Muscle Satellite Cells Revealed with miRNA Interference Technologies.

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