Yu Xiang, Fang Xibi, Gao Ming, Mi Jiaqi, Zhang Xiuqi, Xia Lixin, Zhao Zhihui, Albrecht Elke, Maak Steffen, Yang Runjun
College of Animal Sciences, Jilin University, Changchun 130062, Jilin Province, China.
College of Agriculture, Guangdong Ocean University, Zhanjiang 524088, Guangdong Province, China.
Animals (Basel). 2020 May 9;10(5):818. doi: 10.3390/ani10050818.
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.
阐明前体脂肪细胞分化机制以及脂肪细胞中的脂肪积累是肉牛育种中的一项重要工作。作为重要的转录后调节因子，微小RNA（miRNA）通过结合靶标mRNA的种子位点参与细胞增殖、分化、凋亡和脂肪代谢。本研究的目的是分离和鉴定牛前体脂肪细胞，并筛选与脂肪生成相关的miRNA。从皮下脂肪组织中分离牛前体脂肪细胞，并诱导其分化为脂肪细胞。通过qRT-PCR（实时定量逆转录PCR）、油红O染色和免疫荧光染色对前体脂肪细胞和脂肪细胞进行验证。提取总RNA用于小RNA测序。测序数据显示，131个miRNA在脂肪细胞中高表达，119个miRNA在前体脂肪细胞中高表达。茎环qPCR（茎环定量实时PCR）结果表明，11个miRNA（miR-149-5p、miR-24-3p、miR-199a-5p、miR-33a等）的表达模式与测序结果一致。根据KEGG通路和基因本体论（GO）分析，多个预测的靶基因与脂质代谢相关。总之，本研究提供了一种分离牛前体脂肪细胞并筛选前体脂肪细胞分化过程中各种差异表达miRNA的方法。
