Effect Of MiR-655-3p On Differentiation Of Brown Adipose Tissue Precursor Somatic Cell Cells In Rabbits

The brown fat content of rabbits reaches the peak before and after birth.Brown fat cells are mainly involved in maintaining body temperature and regulating energy metabolism,increasing the activity of brown fat in young rabbits,and effectively improving the survival rate of young rabbits.micro RNAs are involved in regulating the proliferation and differentiation of adipocytes.In this study,the precursor cells of rabbit brown fat were used as materials,and the effects of miR-655-3p on the maturation and differentiation of rabbit brown fat precursor cells were investigated through the screening of miR-655-3p in the ce RNA network after sequencing by molecular breeding method.It provides some theoretical basis for further study on the regulation of miRNAs on the differentiation of brown fat progenitor cells.The results were as follows:(1)Rectal temperature,scapular brown adipose tissue weight and body weight of female rabbits at 15 days were measured.The results showed that the rectal temperature fluctuation amplitude of offspring rabbits with high brown fat/body weight ratio was lower,indicating that offspring rabbits with high brown fat/body weight ratio had better homeostasis regulation ability of body temperature.The results of q PCR showed that the expressions of miR-655-3p and UCP1,PGC1α,CIDEA,FABP4,C/EBPα and PPARγ were also higher in rabbits with low brown fat/body weight ratio,indicating that the brown fat progenitor cells of rabbits with low brown fat/body weight ratio had better differentiation ability.(2)Transcriptomic gene expression levels in scapular brown adipose tissue of female rabbits at 0 d,15 d,85 d and 2 y at birth were determined.The results showed that the expression of miR-655-3p was downregulated gradually during the natural albinism of brown adipose tissue of domestic rabbits.The results of oil red O staining showed that the flower ring lipid droplets increased gradually,and the mature lipid droplets of brown adipocytes showed significant characteristics.The results of q PCR showed that the expression levels of brown fat marker genes UCP1,PGC1α,CIDEA,FABP4,C/EPPα,and PPARγ increased gradually with the increase of induction days,and reached a peak on the8 th day.The results showed that the experiment successfully isolated rabbit brown fat progenitor cells and established the induced differentiation model.(3)On the 3rd day after transfection with miR-655-3p,miR-655-3p expression levels of the overexpressed group(mimic)were significantly up-regulated compared with those of the control group(NC).UCP1,PGC1α,CIDEA,FABP4,C/EPPα,and PPARγ were also higher than those of the NC group.The expression levels of miR-655-3p in inhibitor group were significantly decreased compared with those in control group(INC),and the expression levels of UCP1,PGC1α,CIDEA,FABP4,C/EPPα,and PPARγ were lower than those in INC group.The results showed that overexpression of miR-655-3p promoted the maturation and differentiation of brown fat progenitor cells,while inhibition of expression resulted in the opposite effect.The results of CCK-8 proliferation test and flow cytometry showed that miR-655-3p significantly inhibited the viability of brown adipocytes and inhibited the proliferation of rabbit brown precursor adipocytes.(4)Illumina sequencing of brown fat progenitor cells overexpressed with miR-655-3p showed 306 significant differential genes(DEGs),of which 302 were down-regulated and 4were up-regulated.There were 436 enriched genes in GO analysis,among which 232 BP,13 MF and 29 CC were significantly enriched DEGs,which were mainly involved in REDOX coenzyme metabolism,G protein-coupled receptor signaling pathway,transmembrane signal receptor activity,signal sensor activity and other processes.There are 210 KEGG enrichment pathways in total,among which the significantly enriched pathways are mainly involved in oocyte maturation,oocyte meiosis and cell cycle mediated by progesterone.In conclusion,miR-655-3p promotes the differentiation of brown fat precursor cells and inhibits the proliferation of brown fat precursor cells in rabbits,which provides a theoretical basis for further exploring the role of miR-655-3p in the maturation and differentiation of brown fat precursor cells and bio thermogenesis.