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Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo
Published on: September 18, 2019
[Experimental study on protective effect of small interfering RNA-induced Atrogin-1 gene silencing on muscle cell
Lei Yuan1, Guo-Hao Wu, Bo Zhang
1Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Objective:
To investigate the protective effect of Atrogin-1 gene silencing via RNA interference technique on a model of muscle cell malnutrition.
Methods:
Sequences of five target Atrogin-1 siRNA and the control were selected and synthesized and cloned to vector pBS-hU6-I and then to vector FG12. The length and rightness of the sequences were confirmed. The recombinant FG12 vectors were cotransfected along with pRSVREV, pMDLg/pRRE and pHCMV-G into 293T cells to package lentivirus particles, with which C2C12 cells were infected. The infected C2C12 cells were cultured and differentiated to form myotubes before TNF-alpha was added to induce malnutrition. Expressed products of Atrogin-1 of myotubes were identified by real time PCR and Western blot methods. Myotubes were observed and photographed directly in culture plate without fixation.
Results:
The length and sequences of inserted DNA were right. Compared with the RNA interferencing group, significant atrophy and upregulated expression of Atrogin-1 of myotubes treated by TNF-alpha was found in the control group.
Conclusion:
Atrogin-1 gene silencing could be used to inhibit malnutrition of muscle cells caused by TNF-alpha. Atrogin-1 could be an ideal target in the treatment of cancer cachexia.
Insights
Silencing the Atrogin-1 gene protects muscle cells from malnutrition induced by TNF-alpha. This suggests Atrogin-1 is a potential therapeutic target for conditions like cancer cachexia.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Muscle cell malnutrition is a significant health concern, often linked to conditions like cancer cachexia.
- Atrogin-1 plays a crucial role in muscle protein degradation and atrophy.
Purpose of the Study:
- To investigate the protective effect of Atrogin-1 gene silencing on muscle cells experiencing malnutrition.
- To assess the efficacy of RNA interference (RNAi) in modulating Atrogin-1 expression.
Main Methods:
- Synthesized and cloned Atrogin-1 siRNA sequences into lentiviral vectors.
- Transfected 293T cells to package lentivirus, then infected C2C12 myotubes.
- Induced muscle cell malnutrition using TNF-alpha and analyzed Atrogin-1 expression via real-time PCR and Western blot.
Main Results:
- Successfully constructed and validated recombinant lentiviral vectors for Atrogin-1 gene silencing.
- TNF-alpha treatment led to significant myotube atrophy and increased Atrogin-1 expression in control groups.
- Atrogin-1 gene silencing significantly inhibited TNF-alpha-induced muscle cell malnutrition.
Conclusions:
- Atrogin-1 gene silencing effectively mitigates muscle cell malnutrition induced by TNF-alpha.
- Targeting Atrogin-1 presents a promising therapeutic strategy for muscle wasting diseases, including cancer cachexia.
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