牛长链非编码RNA MIR221HG调控脂肪生成的分子机制解析

Dissecting the regulatory mechanism of adipocyte differentiation is necessary for the molecular breeding and meat quality improvement of agricultural animals. Previously, we have identified a novel long noncoding RNA-MIR221HG which overlapped with a conserved microRNA, miR-221, and demonstrated that MIR221HG can inhibit adipocyte differentiation. However, it still remains unclear that MIR221HG expression is controlled by which transcription factors and how MIR221HG regulating adipocyte differentiation. Bioinformatic analysis revealed that there are several binding sites of adipogenic transcription factor FOXC1 and KLF5 in the promoter region of MIR221HG and it can function as a competing endogenous RNA (ceRNA) for let-7. Therefore, the objective of this study is to investigate the regulatory mechanism of transcription factor FOXC1 and KLF5 on MIR221HG expression, to uncover the regulatory relationship between MIR221HG and miR-221, and to explore if MIR221HG can act as ceRNA to communicate with and regulate protein-coding gene HMGA2 by direct competition for let-7 binding. Our results will comprehensively elucidate the regulatory mechanism of lncRNA-MIR221HG on adipocyte differentiation at the levels of transcription, post-transcription and signal transduction. Our findings will not only shed light on understanding the mechanisms of adipocyte differentiation, but also provide a new theoretical basis for beef cattle breeding and beef quality improvement.

研究脂肪生成的调控机制对于农业动物的分子育种、肉品质提高等具有重要科学意义。申请人前期鉴定到一个新的长链非编码RNA MIR221HG，其外显子区编码一微小RNA miR-221，RNAi试验表明MIR221HG抑制牛的脂肪细胞分化。但MIR221HG表达受上游哪些转录因子调控，以及其通过何种机制调控脂肪细胞分化尚不清楚。分析显示MIR221HG启动子区存在脂分化相关转录因子FOXC1和KLF5的众多结合位点，并且能通过竞争性结合let-7行使功能。因此，本项目旨在解析FOXC1和KLF5调控MIR221HG转录的分子机制，阐明MIR221HG与miR-221的调控关系，构建MIR221HG与其作用靶标let-7、HMGA2之间的互作网络，从转录、转录后、信号转导等层次深入揭示MIR221HG调控脂肪生成的机制，填补牛脂肪发育研究领域的一些不足，为肉牛的分子育种和优质高效生产提供新的思路。

脂肪生成是由一系列转录因子参与的复杂而精细的程序化调控过程，研究脂肪生成的调控机制对于农业动物进行精准的分子育种、提高肉品质等具有重要科学意义。我们在之前的研究中鉴定到一个新的lncRNA，它在牛脂肪细胞分化前后差异表达，由于其与miR-221在基因组上位置重叠，将其命名为miR-221 host gene（MIR221HG）。cDNA末端快速扩增试验表明MIR221HG全长1,064 nt，位于牛的X染色体上，是一个单外显子lncRNA；生物信息学分析表明MIR221HG是一个真正的lncRNA；核质分离结合半定量和定量PCR试验表明MIR221HG主要在细胞核中表达；油红O染色和实时荧光定量PCR分析表明，沉默MIR221HG导致脂肪细胞脂滴数量增加，脂肪细胞分化标志基因PPARγ，C/EBPα和FABP4表达量极显著上调，提示MIR221HG对牛的脂肪细胞分化具有重要的调控作用。试验机理分析表明MIR221HG与miR-221独立行使功能，且MIR221HG的转录激活受转录因子FOXC1调控。本项目为理解脂肪生成的调控机制、肉牛精准育种和优质高效生产提供了新的思路。