MiR-132-3p Targets UCP2 Regulates Differentiation Of Sheep Preadipocytes

The sheep is a ruminant mammal typically kept as livestock.As an important indicator of meat quality,the content of fat in carcass is the focus of attention.Decreasing content of fat is an important breeding goal for mutton sheep.Uncoupling protein UCP2 is a member of the UCPs family.It is also one of the key proteins that promote fat decomposition.When the body begins to use adipose tissue as the main energy source,the level of fatty acids and ketone bodies in the blood increases,fatty acids would promote the expression of UCP2 mRNA,and lipases are activated.MicroRNA(miRNA)has also become a research hotspot in recent years by regulating the effect of functional genes on sheep meat quality.miRNA mainly reduces the mRNA expression of the target gene or degrades the mRNA through complete and incomplete binding to the target gene mRNA.Cultivating and inducing differentiation of sheep precursor adipocytes is an effective means to study miRNA regulation of sheep fat metabolism.Therefore,identification of sheep precursor adipocytes and the influencing factors in the study of sheep adipocyte differentiation are necessary conditions for follow-up experiments.Insulin is an indispensable additive in the differentiation of sheep preadipocytes and plays an important regulatory role in the differentiation process.The aim of this study was to find out the mechanism of miR-132-3p regulating the expression of UCP2 gene in sheep preadipocytes.The differentiation potential of ovine preadipocytes was identified by the expression of oil red O staining and adipocyte differentiation marker genes,respectively.Simultaneously,sheep preadipocytes were treated with different concentrations of insulin for different lengths of time to determine the condition of insulin resistance in sheep preadipocytes and the response to different concentrations of insulin during differentiation of sheep preadipocytes.At different stages of differentiation,cell RNA was extracted and RT-qPCR was used to detect the expression levels of miR-132-3p and UCP2 during the differentiation process of ovine preadipocytes;using three different bioinformatics software to predict miRNAs that can bind to UCP2,and obtain a consensus predicted miRNA that can potentially bind to UCP2;using different concentrations of insulin culture medium for sheep precursor adipocytes treated for different lengths of time to determine the condition of insulin resistance in sheep preadipocytes and response to different concentrations of insulin during differentiation of sheep preadipocytes;construct UCP2 3'-UTR dual-luciferin reporter vector pmirGLO-UCP2-3'-UTR transforms Trans 5a competent cells,extracts and purifies recombinant plasmids;uses double enzyme digestion technology,bacterial liquid PCR technology,and sequencing technology for pmirGLO-UCP2-'-UTR Double fluorescein reporter vectors were identified;mimics of pmirGLO-UCP2-3 '-UTR and miR-132-3p were co-transfected into HEK-293T cells.The relative fluorescence activity was measured to verify the target relationship between miR-132-3p and UCP2;then the mimics of miR-132-3p were overexpressed in sheep preadipocytes and a negative control NC group was established to extract total RNA and total protein.RT-qPCR and Western blotting techniques were used to detect the expression of UCP2 mRNA and protein after over-expression,and simultaneously detect the expression level of the marker gene in sheep preadipocyte differentiation.(1)It was found that the expression of the marker gene gradually increased in the process of inducing differentiation of sheep preadipocytes.The more and more lipid droplets,the larger and larger.After oil red O staining,the cell outline is clear,the red lipid droplets can be seen in the cytoplasm,indicating that the cells have the potential to differentiate into mature sheep fat cells.By adding different concentrations of insulin,it was found that under the conditions of 10-9 mM and 10-8 mM insulin,the cell's glucose uptake increased with the increase of insulin concentration.Under 10-7 mM insulin culture conditions,cells responded to the stimulation of insulin in 24-48 h.With the increase of treatment time,the absorption of glucose increased accordingly.After treatment for 48 h,the cells Can not make a normal response to insulin stimulation,glucose absorption did not change significantly.After 10-6 mM insulin treatment,the cell activity began to decrease,and the cell death rate increased.It was demonstrated that sheep preadipocytes were dose-dependent in 10-9 mM,10-8 mM insulin-producing conditions,and insulin resistance was observed after 10-7 mM insulin treatment for 48 h.(2)Bioinformatics software predicts that miR-132-3p and miR-212-3p are miRNAs that can bind to UCP2,and can be clearly seen on the agarose gel electrophoresis map of the double-enzyme digested plasmid and the bacterial liquid PCR.The presence of the target fragment indicated that the recombinant plasmid contains the UCP2 3'-UTR target fragment;the relative fluorescence activity of the mimics group detected after co-transfection with the recombinant plasmid and miR-132-3p was significantly lower than that of the NC group(P<0.05).The relative fluorescence activity of the Blank group directly demonstrated that miR-132-3p can bind to the UCP2 3'-UTR,indicating that there is a target relationship between miR-132-3p and UCP2.(3)The expression levels of miR-132-3p and UCP2 genes in sheep preadipocyte differentiation were negatively correlated in the early and middle stages of cell differentiation.After overexpression of miR-132-3p,the results of RT-qPCR showed that the expression of UCP2 mRNA in the overexpression group was significantly lower than that in the negative control group,indicating that miR-132-3p is effective against UCP2.The mRNA level was negatively regulated;Western blot showed that UCP2 mRNA expression was significantly lower in the overexpression group(P<0.05)than in the negative control group,indicating that miR-132-3p also negatively regulates the protein level of UCP2.