Transcriptome Analysis of Potential miRNA Involved in Adipogenic Differentiation of C2C12 Myoblasts

>Emerging evidence indicates that microRNA (miRNA) plays an important role in adipogenesis and disease pathogenesis. To investigate the miRNA involved in regulating different periods of adipogenesis, we performed a comprehensive study on microRNAome during the stimulation of adipogenesis by an adipogenic differentiation cocktail in C2C12 myoblasts at 0, 2, 4, and 7 days using the Solexa sequencing technology. In this study, we identified 52 differentially expressed (DE) miRNA. Functional annotation indicated that the target genes of DE miRNA were mostly enriched in adipogenic transdifferentiation and fat metabolism-related pathways, including Wnt, mitogen-activated protein kinase (MAPK), and insulin signaling. The insulin signaling pathway was further analyzed for its close connections to Wnt and MAPK signaling pathways as well as for the containing of thymoma viral proto-oncogene-3 (Akt3) and glycogen synthase kinase-3 beta (Gsk3b), which were both target genes predicted by most DE miRNA. CLIP-seq (crosslinking-immunprecipitation and high-throughput sequencing) data showed that Akt3 was targeted by seven DE miRNA, including five upregulated (miR-203-3p, miR-181c-5p, miR-322-5p, miR-351-5p, and miR-181a-5p) and two downregulated (miR-15b-5p and miR-17-5p) ones. Likewise, Gsk3b was targeted by six DE miRNA, including four upregulated (miR-199a-3p, miR-126-5p, miR-26a-5p, and miR-101a-3p) and two downregulated (miR-150-5p and miR-140-3p) ones. Moreover, Akt3 could regulate the key transcription factor (TF) Foxo1, targeted by two downregulated miRNA (miR-96-5p and miR-183-5p). The expression levels of Akt3 and Gsk3b were downregulated, and TF Foxo1, which worked on the transcription axis of Pgc1a-Pparα-Rxrg-Pparγ to regulate adipogenesis, was upregulated. In conclusion, DE miRNA stimulated the adipogenesis of C2C12 myoblasts through the targeted insulin signaling pathway involving the genes Akt3, Gsk3b, and TF Foxo1.