Effects Of Voluntary Exercise And Caloric Restriction On Skeletal Muscle Autophagy-related Microrna Expression Of Mice

Autophagy is a universal and highly conserved process involving degradation of cellular organelles in eukaryotic cells. It plays an important role in cell growth and differentiation and is involved in the pathological process of a variety of diseases such as cancer, neural degenerative diseases and conditions related to autophagy deficiency, such as myopathy and aging etc. Notably, the role of autophagy in maintaining the protein balance of skeletal muscle, the removal of metabolic product, as well as the reconstruction of skeletal muscle cells has received more and more attentions.MicroRNAs (miRNAs, also called small RNA) is a class of non-coding small single-stranded RNAs in eukaryotic cells with the length of21to25nucleotides. MicroRNAs regulate gene expression at post-transcriptional level by pairing with target mRNAs, resulting in mRNA degradation or the inhibition of mRNA translation. miRNAs functionally participate in the regulation of a variety of cellular activities, including cell proliferation, differentiation, apoptosis and autophagy. miRNAs are also involved in cellular metabolism such as glucose metabolism and lipid metabolism, and are closely associated with many diseases, including type2diabetes, alzheimer’s disease, cancers, etc. A number of studies have shown that miRNAs play a crucial role in regulating autophagic pathways in various tissues and cells.Many studies have shown that exercise can activate autophagy, and that moderate level of autophagy is very important to maintain skeletal muscle homeostasis. Similarly, moderate caloric restriction (CR) can attenuate the reduction of skeletal muscle autophagy caused by aging, which possibly explains why CR could slow down aging-caused muscle damage and apoptosis. However, whether the effects of exercise and CR on autophagy are associated with miRNAs expressions is still unknown. In the current study, voluntary wheel running and CR model were set up to observe the regulation of skeletal muscle autophagy by miRNAs. We hope to provide new insight for the quality control of skeletal muscle from the perspective of skeletal muscle autophagy activation.Objective:1) To verify the p53-miR-34positive feedback loop in C2C12cells and provide theories for the subsequent animal experiments.2) To explore the role of p53miR-34positive feedback loop in autophagy, thus providing new insight for the quality control of skeletal muscle from the perspective of skeletal muscle autophagy activation.Methods:1) Mice C2C12cell line was transfected with miRNAs mimic, and was divided into control group (C) and experimental group (T).2)18ICR8-week mice were randomly assigned to control group (N), caloric restriction group (CR) and voluntary exercise group (E) with6in each. Group N was fed with food ad libitum. Group E was subjected for8weeks of voluntary wheel running. Group CR was fed according to a CR dietary regimen. The gastrocnemius muscle of all mice were collected at the end of the intervention period. RT-PCR and western blot were performed to examine the mRNA and protein expression levels.Results:1) In the cell experiments, the miR-34expression level in group T was significantly increased compared to group C (p<0.01). The mRNA expression of BCL-2in group T was significantly lower than in group C (p<0.05). The mRNA expressions of LC3b, P62and ULK1in group T were significantly higher than in group C (p<0.01).2) In the animal experiments, the body weight of group CR was significantly lower than that of groups C and E since the third week (p<0.01). The mRNA expressions of Atg13and Atg7of group CR was significantly higher than group N (p<0.05). The protein levels of p53and Sirtl in group CR were significantly higher (p<0.05) and lower (p<0.05) than in group N, respectively. As regard to the effect of exercise, the mRNA expression levels of p53, miR-34a, LC3, p62, Atgl3, Atg5and Atg7were significantly higher than in group N (p<0.05). The protein levels of p53and LC3b were significantly higher than in group N (p<0.01), while the protein levels of Sirtl and Bcl-2were significantly lower than in group N (p<0.05).Conclusion:1) miR-34a overexpression can cause changes in skeletal muscle autophagy related gene expression.2) With the intervention of voluntary wheel running and CR, the mice skeletal muscle autophagy level rises and is regulated by the p53-miR-34a positive feedback loop.