Isolation and identification of bovine preadipocytes and screening of miRNAs associated with adipogenesis

Introduction: In the breeding of beef cattle, the adipogenesis is a significant process to fat deposition. Thus isolating and identifying bovine preadipocytes in vitro is a basis for investigating mechanism of adipogenesis. As important transcriptional regulators, miRNAs play a pivotal role in adipogenesis. Materials and methods: Bovine preadipocytes were isolated from subcutaneous fatty tissue and induced to differentiate into adipocytes. The verification of preadipocytes and adipocytes was performed by qPCR, Oil Red O stain and triglycerides detection. Total RNAs were extracted from bovine preadipocytes and adipocytes and proceeded small-RNA sequencing. KEGG pathway analysis and GO analysis were used to screen differently expressed miRNAs associated with adipogenesis. Results: The result showed that more lipid drops in induced group were observed and stained red compared with control group. The expression levels of key genes (ppary, c/ebpa, fabp4, fasn and leptin) associated with adipogenesis were increased. Dlk1, as a marker gene of preadipocytes, was scarcely expressed in adipocytes. The content of cellar triglycerides detected in adipocytes was significantly upgraded. 131 miRNAs were found to be highly expressed in bovine adipocytes, and 119 miRNAs were highly expressed in bovine preadipocytes. KEGG pathway analysis showed that 4.76% of the differentially expressed genes were enriched in lipid metabolism. Discussion and conclusion: Previous studies have found that some miRNAs could regulate lipid metabolism. But few studies have explored the relationship between miRNAs and adipogenesis. In our study, we characterized the miRNAs associated with adipogenesis in bovine preadipocytes. The expression profiles of 11 miRNAs verified by q-PCR, including miR-149-5p, miR-199a-3p, miR-199a-5p, miR-148a and miR-449a were the same to sequencing data. Based on the KEGG pathway analysis, the predicted target genes were mainly enriched in signal transduction, catabolism and lipid metabolism. This work contributes to existing knowledge by providing an understanding of bovine adipogenesis at the molecular level.