Effect And Mechanism Of Bovine MiR-378in Preadipocyte Differentiation

Adipose tissue is crucial for both energy storageand metabolismregulations. Also,the proportion of the adipose tissue deposition in the body of animals and meat is animportant topic in genetics and breeding research. The animal fat generation isregulated by many factors, such as genes, enzymes and hormones. Previousresearches have reported that microRNAs(miRNAs) could regulate the metabolism ofadipocyte by combining with the specific sites of target genes which are associatedwith adipocyte differentiation to degradate its mRNAs or proteins. Some reports haveshown that, miR-378can promote the differentiation of3T3-L1cells, but theregulation mechanism of miR-378in primary preadipocytes differentiation is unclear.In order to identify the functions of miR-378in primary bovine preadipocytedifferentiation processes,we tested the expression of miR-378in different tissues ofbovine, The results showed that the expression level of miR-378is not only high inpreadipocytes, but also in the heart, spleen and small intestine.After the induction ofpreadipocyte differentiation, the level of miR-378in the differentiated lipid dropletwas up-regulated significantly, which reveals the expression pattern of miR-378indifferentiation process of bovine tissues and preadipocyte.miR-378plays a role in regulating the target gene in preadipocyte cells, wecombing bioinformatics and in vitro dual luciferase report system, we found thatmiR-378could inhibit the E2F2and RANBP103’-UTR’s dual luciferase reporterrecombinant plasmid expressing luciferase activity. When E2F2and RANBP10mutant recombinant plasmids with miR-378mimics were transfected into NIH3T3cell line,inhibition of miR-378on E2F2luciferase activity was decreased from70%to30%, RANBP10luciferase activity was decreased from75%to10%. The result shows that miR-378regulats E2F2and RANBP10by binding seed zones as its targetsites.To further analyze the mechanism of miR-378for post transcriptional regulationof target gene,we transfected miR-378into the preadipocyte,the results showed thatthe expression level of target genes mRNA unchanged significantly, while the proteinexpression level was down regulated. miR-378regulats E2F2and RANBP10throughinhibiting their mRNA translation process rather than transcription process.miR-378was transfected into the preadipocyte to induce the differentiation, the lipid dropletcontent and volume was up-regulated, triglyceride accumulation was increased, andsome factors associated with the differentiation and transcription of preadipocytetranscription express differences in miR-378transfection induced group.In summary, the study highlights the high expression of miR-378in preadipocyte.And expression of which is upregulated in the later stage of differentiation. Wescreened and identified miR-378target genes, E2F2and RANBP10,and promotingbovine preadipocyte differentiation by regulating its target gene. Therefore, our studyprovides a theoretical basis for further study on bovine miRNA’s effects in fatdeposition, fleshy polish the molecular regulation mechanism.