Abstract: The research team previously utilized miRNA sequencing technology to identify a differentially expressed and highly conserved miRNA, miR-196b-5p, in the soleus and longissimus dorsi muscles of large white pigs. Previous studies have demonstrated that miR-196b-5p promotes the proliferation and differentiation of C2C12 myoblasts, its role in myofiber type conversion remains unclear. To explore the effects and regulatory mechanisms of miR-196b-5p on skeletal muscle fiber type conversion, this study used mice as experimental subjects and systematically analyzed the regulatory role of miR-196b-5p by injecting AAV-miR-196-5p and AAV-empty into mice muscles. The statistical results showed that AAV-miR-196-5p administration did not significantly influence the body weight, feed intake, or tissue weight of the mice （P>0.05）, confirming no interference with normal growth. H&E staining results revealed that miR-196b-5p overexpression significantly enlarged fiber cross-sectional areas in both tibialis anterior muscle （TA） and gastrocnemius muscle （GAS） （P<0.05）. Real-time fluorescence quantitative PCR results demonstrated that miR-196b-5p overexpression downregulated the slow-twitch muscle fiber marker genes,MyHC ⅠandMyHC Ⅱa（P<0.05） but upregulated the fast-twitch muscle fiber marker gene,MyHC Ⅱb（P<0.05）. Additionally, the overexpression of miR-196b-5p significantly reduced the mRNA expression levels of mitochondrial oxidative phosphorylation related genes （COX5B,COX6C,COX7A1, andCOX8B） （P<0.01）, suggesting that miR-196b-5p may inhibit the oxidative metabolism of skeletal muscle. KEGG enrichment analysis of the target genes of miR-196b-5p revealed that these genes were mainly enriched in the p38 mitogen-activated protein kinase （p38-MAPK） pathway and the AMP-activated protein kinase （AMPK） pathway. Western blot showed that the overexpression of miR-196b-5p significantly reduced the protein phosphorylation level of p38-MAPK （P<0.05） while significantly increasing the protein phosphorylation level of AMPK （P<0.05）. Furthermore, overexpression of miR-196b-5p significantly reduced the protein expression level of the slow-twitch muscle fiber marker gene MyHCⅠ （P<0.05）. These findings suggested that miR-196b-5p played a crucial role in skeletal muscle fiber type conversion via AMPK activation and p38-MAPK inhibition, thereby suppressing oxidative metabolism, reducing the formation of slow-twitch muscle fibers, and promoting the formation of fast-twitch muscle fibers.

Key words: miR-196b-5p, skeletal muscle, myofiber type, p38-MAPK pathway, AMPK pathway