The Functional Research Of MiR-103Towards Porcine Adipocyte Differentiation

MicroRNAs are small non-coding RNA that regulate their target genes atpost-transcription level (Wightman et al.1993；Reinhart et al.2000), they exert regulatoryfunction in nearly every kinds of biological processes, e.g. cancer, aging, metabolism, celldifferentiation ect.(Alvarez-Garcia and Miska2005). Recently, increasing number ofarticles demonstrate that microRNAs also play an important part in the molecular networkof adipogenesis. In human and mouse, tons of adipogenesis regulate microRNAs wereidentified, e.g. miR-17-92and miR-143were proved to promote preadipocytedifferentiation (Wang et al.2008; Esau et al.2004), while miR-27a,miR-27b and let-7werereported to inhibit preadipocyte differentiation (Kim SY et al.2010; Karbiener M et al.2009;Sun et al.2009). However, in porcine, similar researches are still in the beginning. Sostudying of the function and mechanism of microRNA in porcine preadipocytedifferentiation is really necessary. Our laboratory took out solixa sequencing with thepreadipose of mature porcine and piglet before hand, and found that miR-103had a notabledifferent expression level in these two tissues, which prefigure that miR-103may participatein preadipocyte differentiation, so we regard miR-103as our study object. This studyprovides new reference for the construction of microRNA regulatory network ofpreadipocyte differentiation, also present theoretical foundation for genetic breedingresearch, the main results of this paper are listed as below:1. The construction of miR-103adenovirus over-expression vectorWe Used pre-miR-103sequence containing vector pGenesil-1.2as the template toamplify pre-miR-103sequence, and connected with pAdTrack-CMV vector, thenrecombinant with backbone plasmid, in this way, we finished the construction ofpAd-miR-103plasmid vector. Used Lipofecta mineTM2000to package adenovirus, andthen proliferated and concentrated it, the adenovirus that generated was calledAd-pre-miR-103. Amplified Ad-pre-miR-103to infect porcine preadipocyte, and theinfection efficiency reached nearly100%;48h after infection, we found the expression ofmiR-103increased3.7folds. 2. The effect of miR-103towards porcine preadipocyte differentiation.Infected porcine preadipocyte with Ad-pre-miR-103and empty adenovirus, the latergroup were used as control. Collected cells after induced for0,2,4,6,8d respectively, thenextracted cell total RNA and protein, after real-time PCR and western blotting we foundpreadipocyte infected by Ad-miR-103have an notable elevated mRNA level of adipocytemarker gene PPARγ,aP2and elevated protein level of aP2; in the8th day after inducing,there were a lot of lipid droplets flocked together, when stained with oil red O, we found thegroup infected by Ad-miR-103have greater amount of lipid droplets than empty adenovirusinfected group. The above-mentioned demonstrate that miR-103can promote adipocytedifferentiation.3. The prediction of miR-103target geneThe way MicroRNAs exerting their function, after all, is through regulating their targetgenes, so unveiling microRNA target gene is the key method to clarify their mechanism.MicroRNAs inhibit target gene expression post-transcriptionally, mainly through theuncomplemented pairing with target gene3′UTR. This experiment amplified Pictar,TargetScan,miRanda etc. target gene prediction software distinguished miR-103target gene—RAI14,MEF2D,ANK1and FGF2, which indicate that the adipogenesis simulativefunction of miR-103is probably through the regulation of these genes.