2023, Vol. 27 ›› Issue (5): 732-737

Differential expression profiles of microRNAs in muscle tissue of denervated skeletal muscle atrophy rats

Liu Hongwen1, 2, Li Jiao3, Xu Wenhao3, Nie Hua1, Liu Shaojiang1, Xu Jie2, Yin Li1, 3   

Abstract: BACKGROUND: There is no effective treatment for denervated skeletal muscle atrophy with poor prognosis, and moreover, the expression profiles of microRNA in vivo are unclear.
OBJECTIVE: To analyze the microRNA expression profiles and the changes of transforming growth factor-β1/Smad3 signaling pathway in denervated skeletal muscle atrophy rats using high-throughput sequencing technology and to explore the role and mechanism of microRNA in skeletal muscle atrophy.
METHODS: Twelve Sprague-Dawley rats were randomized into two groups: experimental group and control group. Animal models of denervated muscle atrophy were established using truncation of the sciatic nerve in the experimental group. In the control group, only the sciatic nerve was exposed followed by suturing. The bilateral gastrocnemius muscles were completely removed to calculate the muscle wet weight ratio. Atrophy and fibrosis were observed by hematoxylin-eosin staining and the cross-sectional area of the muscle was determined. Illumina HiSeq 2500 sequencing technology was used to identify the microRNA expression profiles in skeletal muscle tissue. In combination with volcano plot, hierarchical clustering map, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway, differentially expressed genes involved in skeletal muscle metabolism were analyzed. Real-time fluorescent quantitative PCR was used to verify the differential expression of candidate genes in muscle tissue and western blot was used to detect the expression of transforming growth factor-β1 and p-Smad3 proteins.
RESULTS AND CONCLUSION: Compared with the control group, the muscle wet weight ratio and cross-sectional area were significantly lower in the experimental group (P < 0.05), and the modeling results were in line with expectations. A total of 1 249 differentially expressed microRNAs were identified in the two groups (P < 0.05; | log2 Fold Change | > 0.0). There were 14 microRNAs with significant differential expression, of which 2 were up-regulated and 12 were down-regulated. The results of biological function and pathway analysis indicated that miR-1247-3p, miR-132-5p, miR-21-3p, miR-363-3p, and
miR-451-5p with significant differential expression were significantly enriched in cell proliferation, differentiation, apoptosis and other biological processes as well as transforming growth factor-β signaling pathway. The results of real-time fluorescent quantitative PCR showed that the expression of miR-21-3p in the gastrocnemius muscle tissue of the experimental group was significantly lower (P < 0.001), which was consistent with the trend of the high-throughput sequencing results. Western blot results showed that the protein expression levels of transforming growth factor-β1 and p-Smad3 were significantly higher in the experimental group than the control group (P < 0.05). To conclude, microRNA plays a key role in the physiological and pathological processes of skeletal muscle atrophy. miR-21-3p may play a role in skeletal muscle cell metabolism by activating the biological activity of transforming growth factor-β1/Smad3 signaling pathway.
Key words: denervation, skeletal muscle atrophy, high-throughput sequencing technology, microRNA, transforming growth factor-β1, Smad3