The Role And Mechanism Of MiR-10a-5p In The Proliferationand Differentiation Of Preadipocytes

Micro-RNAs,a class of non-coding RNAs,regulate the expression of genes by inducing the cleavage of messenger RNA(mRNA)or inhibition of protein translation,thereby participating in various biological processes of the organism.In recent years,a number of papers have shown changes of microRNA expression in obese individuals versus normal individuals,and these changes seem to help us understand the underlying factors of metabolic disorders in this patient population.In the process of livestock breeding,the phenomenon of over-fertilization often occurs.On the one hand,it has an impact on livestock breeding,which reduces the speed of genetic breeding.On the other hand,it also affects the slaughter value of meat livestock,thereby reducing its economic value.The study found that miRNA,as a post-transcriptional level regulator,plays an important regulatory role in the differentiation of adipocytes.According to the preliminary experiments,it was found that miR-10a-5p was expressed in all tissues,but the expression level was the lowest in subcutaneous adipose tissue,and miR-10a-5p is highly conserved among different species such as human,mouse,rat and pig.According to the sequencing results,differentially expressed miR-10a-5p was screened in the longissimus dorsi(Ld)and semitendinosus(Sd)of pigs,and its expression level was significantly higher in the Ld than in the Sd(P <0.01),and Our laboratory has reported that the content of preadipocyte in the Ld is higher than that of the Sd.It is predicted that there is a regulatory relationship between the adipogenic differentiation of subcutaneous preadipocyte.Based on this,in this study,C57 mice were used as experimental animal models,and it is hoped that this will explore the biological functions and potential mechanisms of miR-10a-5p in the proliferation and differentiation of subcutaneous preadipocyte.The subcutaneous preadipocyte was isolated and cultured in vitro,were transfected with miR-10a-5p agomir in the proliferative phase and the differentiated phase,to explore their effects on the proliferation and differentiation of subcutaneous preadipocytes.Next,the proliferative target gene Map2k6 3' UTR and the differentiated target gene Fasn 3' UTR dual luciferase reporter vector were constructed to verify their direct targeting relationship with miR-10a-5p.The above studies have the following main results:1.miR-10a-5p promotes the proliferation of subcutaneous preadipocytesOverexpression of miR-10a-5p in subcutaneous preadipocytes promoted the proliferation of subcutaneous preadipocytes,and the proliferative-related genes Cyclin D(P < 0.01)and PCNA(P < 0.05)mRNA levels increased significantly,Cyclin E,Cyclin B were also a certain increased;while the mRNA expression levels of the proliferative-associated genes p27 and p21 were significantly decreased(P < 0.05).At the protein level,the expressions of proliferating-related genes Cyclin D(P < 0.01),Cyclin B and PCNA were significantly increased,while the protein expression levels of proliferative-associated genes p27 and p21 were significantly decreased(P < 0.05);Flow cytometric analysis showed that the cells were more transformed into the interphase S phase,and the ability to replicate DNA molecules was enhanced.The results of EdU staining and CCK8 further confirmed the promotion of miR-10a-5p in the proliferative phase of subcutaneous preadipocytes.2.miR-10a-5p targets Map2k6 during the proliferative phaseMultiple target gene online software predicted Map2k6 as a candidate target gene of miR-10a-5p,and the temporal expression of Map2k6 in the proliferative phase was opposite to that of miR-10a-5p,constructing Map2k6 3'UTR dual luciferase reporter vector,It was confirmed by enzyme activity assay that Map2k6 is a target gene of miR-10a-5p.3.miR-10a-5p inhibits differentiation of subcutaneous preadipocytesAfter overexpression of miR-10a-5p,the expression levels of PPAR? and aP2 mRNAs in adipogenic differentiation of subcutaneous preadipocytes were significantly down-regulated(P < 0.05).At the protein level,the expression levels of PPAR?,aP2 and adipoQ proteins decreased significantly,and the p-AKT,a key signaling pathway for adipogenesis,also decreased significantly(P < 0.01).Moreover,oil red O staining and its quantification showed that transfection of miR-10a-5p significantly reduced the accumulation of lipid droplets during differentiation(P < 0.05).This indicates that overexpression of miR-10a-5p can inhibit the differentiation of subcutaneous precursor adipocytes.4.miR-10a-5p targets Fasn during differentiationAccording to the software prediction and combined with the phenotype of inhibition,Fasn was used as a candidate gene for miR-10a-5p to regulate the adipogenic phase.It was found that the temporal expression of Fasn was opposite to that of miR-10a-5p.Next,a Fasn 3'UTR dual luciferase reporter vector was constructed,and Fasn was identified as a target gene of miR-10a-5p by enzyme activity assay.Taken together,this study found that miR-10a-5p is underexpressed in subcutaneous fat.In vitro culture,overexpression of miR-10a-5p significantly promoted the proliferation of subcutaneous preadipocytes and significantly inhibited subcutaneous preadipocytes differentiation.It indicates that miR-10a-5p is involved in the regulation of lipogenesis,which not only enriches the research network of miRNA,but also provides a theoretical basis for further study on the molecular mechanism of fat deposition traits in livestock.