Isolation and Identification of Bovine Preadipocytes and Screening of MicroRNAs Associated with Adipogenesis

SIMPLE SUMMARY: Promoting fat deposition in beef cattle has been a focus of modern animal breeding research. However, previous researchers have not examined the mechanism of adipogenesis in much detail. MicroRNAs (miRNAs) are small noncoding RNAs that play a pivotal role in adipogenesis. In this study, to explore the molecular regulatory mechanism of adipocyte differentiation and formation, bovine preadipocytes were isolated and induced into adipocytes, and then the expression patterns of miRNAs between preadipocytes and adipocytes were detected through RNA sequencing. Deep sequence analysis has identified 78, 71, and 48 novel miRNAs and 497, 491, and 524 known miRNAs in the preadipocytes, and 44, 54, and 47 novel miRNAs and 519, 522, and 504 known miRNAs in the adipocytes. Among the annotated miRNAs, 131 bovine miRNAs were upregulated in adipocytes, and 119 bovine miRNAs were downregulated in adipocytes, such as bta-miR-3604, bta-miR-23b-3p, bta-miR-26a, and bta-miR-129-3p. Bovine target gene prediction results of these miRNAs show that numerous genes are associated with lipid metabolism. These results can provide both technical support and a research basis for promoting bovine adipocyte fat deposition. ABSTRACT: The elucidation of the mechanisms of preadipocyte differentiation and fat accumulation in adipocytes is a major work in beef cattle breeding. As important post-transcriptional regulators, microRNAs (miRNAs) take part in cell proliferation, differentiation, apoptosis, and fat metabolism through binding seed sites of targeting mRNAs. The aim of this study was to isolate and identify bovine preadipocytes and screen miRNAs associated with adipogenesis. Bovine preadipocytes were isolated from subcutaneous fatty tissue and induced to differentiate into adipocytes. Verification of preadipocytes and adipocytes was performed by qRT-PCR (real-time quantitative reverse transcription PCR), Oil Red O staining, and immunofluorescence staining. Total RNA was extracted for small RNA sequencing. The sequencing data showed that 131 miRNAs were highly expressed in adipocytes, and 119 miRNAs were highly expressed in preadipocytes. Stem–loop qPCR (stem–loop quantitative real-time PCR) results showed that the expression patterns of 11 miRNAs were consistent with the sequencing results (miR-149-5p, miR-24-3p, miR-199a-5p, miR-33a, etc.). According to KEGG pathway and Gene Ontology (GO) analyses, multiple predicted target genes were associated with lipid metabolism. In summary, this study provides a protocol of isolating bovine preadipocytes and screening various differently expressed miRNAs during preadipocyte differentiation.