Regulation Of NR4A1 On The Differentiation Of Intramuscular Preadipocytes In Goat

The quality of lamb is affected by many factors,among which the content of intramuscular fat is an important factor affecting the quality of lamb.As a transcription factor,Nuclear Receptor Subfamily 4 Group A Member 1(NR4A1)binds to downstream target genes plays an important role in cell proliferation and differentiation through signaling pathways.However,there is no report on the differentiation of goat intramuscular preadipocytes.Therefore,this paper explores the regulation of NR4A1 on goat intramuscular preadipocyte differentiation.So,this study uses RT-PCR,Real-time Quantitative PCR(q PCR),vector construction,cell culture,overexpression,RNA interference,RNA-Seq and other analysis and biological methods to clarify the regulation and possible mechanism of NR4A1 on goat adipocytes differentiation.(1)Goat NR4A1 gene cloning and tissue expression characteristicsThe ORF sequence of goat NR4A1 was successfully cloned in this study.The length of the goat NR4A1 is 1797 bp,which encodes 598 amino acids.It has a highly conserved zinc finger structure and a DNA structural site;NR4A1 is widely expressed in 10 tissues including heart,liver,and spleen,the highest expression is in goat longissimus dorsi muscle(P<0.05),followed by lung(P<0.01).(2)The effect of NR4A1 on the differentiation of goat preadipocytesBased on the obtained goat NR4A1 sequence,an overexpression vector(pc DNA3.1-NR4A1)was constructed using double enzyme digestion method,and NR4A1 si RNA was synthesized.After overexpression of NR4A1 in goat intramuscular adipocytes,the accumulation of intracellular lipid droplets increased,and the relative expression levels of AP2 、 SREBP1 、 ACC 、 GPAM and DGAT2 increased significantly(P<0.05),the relative expression level of Pref-1 and HSL was significantly reduced(P<0.05).After interfering with NR4A1 in goat intramuscular adipocytes,intracellular lipid droplets were significantly reduced,and the relative expression levels of LPL、CEBPα、CEBPβ、ACC and DGAT2 were significantly decreased(P<0.05),the relative expression levels of Pref-1 and HSL were significantly increased(P<0.05).(3)NR4A1 regulates goat intramuscular preadipocytes differentiation through PI3K-Akt signaling pathwayAfter overexpressing NR4A1 in goat intramuscular preadipocytes,a total of 690 differentially regulated expression genes were obtained through high-throughput sequencing technology,of which 446 were up-regulated and 244 were down-regulated.KEGG enrichment found that differential genes were significantly enriched in 86 pathways,among the pathways related to adipocyte differentiation,there are more genes enriched in the PI3K-Akt signaling pathway,and NR4A1 gene is also enriched in the PI3K-Akt signaling pathway.Adding PI3K-Akt signaling pathway inhibitor LY249002 after overexpression of NR4A1 prevents overexpression of NR4A1 from promoting the differentiation of intramuscular preadipocytes in goats,and may reduce the relative expression levels of C/EBPα、AP2、SREBP1、C/EBPβ、ACC、GPAM、DGAT1、DGAT2 and ATGL are achieved.The above results indicate that NR4A1 promotes the differentiation of intramuscular and subcutaneous preadipocytes in goat.The results provided important references for elucidating the molecular mechanism for goat meat formation,and it also provided candidate genes for goat molecular breeding.