Isolation and Identification of Bovine Preadipocytes and Screening of MicroRNAs Associated with Adipogenesis
Isolation and Identification of Bovine Preadipocytes and Screening of MicroRNAs Associated with Adipogenesis
复制标题
牛前脂肪细胞的分离鉴定及脂肪生成相关MicroRNA的筛选
作者：
Yu Xiang;Fang Xibi;Gao Ming;Mi Jiaqi;Zhang Xiuqi;Xia Lixin;Zhao Zhihui;Albrecht Elke;Maak Steffen;Yang Runjun
促进肉牛脂肪沉积一直是现代动物育种研究的热点。然而，以前的研究人员并没有详细研究脂肪形成的机制。MicroRNAs （miRNAs）是一种小的非编码rna，在脂肪形成中起着关键作用。本研究为探索脂肪细胞分化形成的分子调控机制，将牛前脂肪细胞分离诱导成脂肪细胞，通过RNA测序检测前脂肪细胞与脂肪细胞之间miRNAs的表达模式。深度序列分析在脂肪前细胞中鉴定出78、71和48个新mirna， 497,491和524个已知mirna；在脂肪细胞中鉴定出44、54和47个新mirna， 519、522和504个已知mirna。在注释的miRNAs中，131个牛miRNAs在脂肪细胞中上调，119个牛miRNAs在脂肪细胞中下调，如bta-miR-3604、bta-miR-23b-3p、bta-miR-26a和bta-miR-129-3p。这些mirna对牛靶基因的预测结果表明，许多基因与脂质代谢有关。这些结果可为促进牛脂肪细胞脂肪沉积提供技术支持和研究基础。摘要阐明前脂肪细胞分化和脂肪细胞内脂肪积累的机制是肉牛育种中的一项重要工作。microRNAs （miRNAs）作为重要的转录后调节因子，通过结合靶向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脂肪细胞，并筛选与脂肪形成相关的mirna。从牛皮下脂肪组织中分离前脂肪细胞，诱导其向脂肪细胞分化。采用qRT-PCR（实时定量反转录PCR）、油红O染色和免疫荧光染色对前脂肪细胞和脂肪细胞进行验证。提取总RNA进行小RNA测序。测序数据显示，131个mirna在脂肪细胞中高表达，119个mirna在脂肪前细胞中高表达。茎环qPCR （Stem-loop quantitative real-time PCR）结果显示，11个mirna的表达模式与测序结果一致（miR-149-5p、miR-24-3p、miR-199a-5p、miR-33a等）。根据KEGG通路和基因本体（Gene Ontology， GO）分析，多个预测靶基因与脂质代谢相关。综上所述，本研究提供了分离牛前脂肪细胞和筛选前脂肪细胞分化过程中各种不同表达的mirna的方案。
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.