Investigating The Role Of Microrna-191in Porcine Adipogenesis

It has been demonstrated that the maintenance of adipose tissue function depends on the formation capacity of adipocytes. The formation of matured adipocyte consists of the proliferation, commitment and differentiation of mesenchymal stem cells, which are regulated by the cascade process of adipocyte transcription factors, multiple signal pathways and miRNAs.The miRNAs expression profiles of back subcutaneous fat of Rongchang piglets and adult pigs were revealed by Solexa sequencing. The functions of miRNAs showing significant expression differences between piglets and adult pigs such as that of miR-27, miR-196and miR-103has been examined by the former studies, which showed that these miRNAs affected the post-transcription of PPARγ, C/EBPα and ap2, thereby participating in the commitment and differentiation of porcine preadipocytes and3T3-L1cells. To identify the new adipose regulator and obese therapeutic targets, we combined the results of Solexa sequencing with the prediction of miRNAs possessing the potential of regulating adipogenesis. In this article, the functions and mechanisms of microRNA-191during the adipogenesis of swine preadipocytes are clarified.By checking miRbase, we found that the ssc-miR-191is located in the reverse strand of chromsome13, the precursor of ssc-miR-191contains80nucleotides, the matured is23nt, located in the first intron of D-arabitol transcriptional repressor anticodon binding domain containing3(DALR3). Former studies identified that the sequence of miR-191was completely conserved among the vertebrates, and it is located in the intron of DALR3in all vertebrates, the expression of miR-191is accordant to the expression of DALR3. Recent researches showed that miR-191has a function on the tumor formation, it can also regulate the adipogenesis-related genes like C/EBPβ, CDK6and TCF7L and signal pathways like MAPK/ERK, TGF-βand MAPK/JNK, but the function of miR-191in adipogenesis is still unreported.In present study, the expression profile of miR-191during the adipogenesis of porcine preadipocytes was examined by Real time-qPCR. Ad-miR-191and Ad-miR-NTC were transfected into the porcine preadipocyte respectively to indentify the impact of miR-191on the commitment and differentiation of porcine preadipocytes, the difference between Ad-miR-191and Ad-miR-NTC treatments in the lipid synthesizing capacity was detected by oil red O stain, and the expression level of mRNA and protein of adipogenesis-related genes were examined by Real time-qPCR and Western blot respectively. Also, the target genes of miR-191were selected by bioinformatics analyses and the target genes were identified by dual luciferase report system and Western blot.The main results are as follow:1. C/EBPβ, the potential target gene of miR-191, was selected by PicTar and TargetScan, and the result of reverse selection by MicroInspector showed that the combination of miR-191and C/EBPβ possessed the lowest binding free energy.2. The expression level of miR-191in porcine preadipocyte during the adipogenesis was increased, which was consistent with the results of Solexa sequencing.3. The overexpression of miR-191had an inhibited effect on the differentiation of porcine preadipocyte. The proportion of the mature cells decreased, the content of lipid in cell reduced, and the expression levels of the marker genes on differentiation were down-regulated.4. miR-191could bind the3’UTR of C/EBPβ and down-regulate the firefly luciferase activity; miR-191inhibited the protein expression of C/EBPP during the adipogenesis.In conclusion, miR-191has a negative effect on the differentiation of preadipocyte, mainly by regulating adipogenesis key gene C/EBPβ. These results lay the foundation for further studies on the mechanism of the regulating of adipogenesis by miR-191.