Identification Of MicroRNAs Profiles And Functional Analysis Of MiR-34c In Porcine Intramuscular Adipocyte Adipogenesis

Intramuscular fat(IMF)content,one of the key factors determining the meat quality,is gaining more attention in the meat production industry.Therefore,from the point of IMF content to improve the pork quality became a hot research topic in animal husbandry.IMF is affected by intrinsic genetic and/or extrinsic nutritional factors.The effect of these factors on IMF could be studied more directly in the cultured intramuscular adipocyte in vitro.MicroRNAs(miRNAs)are a class of short and endogenously initiated non-coding RNAs(19-22 nt),which could post-transcriptionally regulate gene expression.Up to date,multiple miRNAs have been identified in adipose tissue and muscle.However,systemic identification of porcine skeletal muscle miRNAs profile and functional analysis of miRNA in the development of IMF remains still elusive.Also,previous studies showed that the intramuscular adipocyte of semitendinosus muscle(SM)are significantly different from that of the longissimus muscle(LM).In this study,intramuscular adipocyte were isolated from a mixed cell populations according to their differential attaching from SM and LM,respectively.Then the RNAs of cultured intramuscular preadipocyte and mature adipocyte were used to perform high throughout sequencing to screen the differential miRNAs.miR-34-c were selected as one of the candidates from the sequencing analysis.The potential role of miR-34 c and its regulation mechanism in the intramuscular preadipocyte proliferation and differentiation were further explored by overexpression,real-time qPCR,Western blot,double luciferase assay and flow cytometer analysis.The main results are as follows:1.L-IMA(intramuscular preadipocytes from porcine longissimus muscles)has stronger ability of differentiation and lipid accumulation than that of S-IMA(intramuscular preadipocytes from porcine semitendinosus muscles).L-IMA has higher population of mature adipocyte and more TG content.Also,the level of adipogenic genes expression,including PPAR?,SREBP-1,C/EBPs and FABP4 in L-IMA is higher than in S-IMA.These results indicate that the capacity of intramuscular preadipocyte differentiation is different from depots of skeletal muscles.2.Compared to L-IMA,the adipogenic pathway was affected in S-IMA.Phosphorylation of AKT,mTOR and ERK1/2 was downregulated in S-IMA(P < 0.05).3.High throughout sequencing between L-IMA and S-IMA at different differentiation stage(Ld0,L2,L8,S0,S2 and S8)showed that there were 525,521,543,533,552 and 545 of known miRNAs in Ld0,L2,L8,S0,S2 and S8 samples,respectively.The number of new miRNAs was 1428,1295,789,1572,1184 and 1081 in these samples,respectively.4.The differential expression assay showed that 21 miRNAs had similar expression pattern.9 of them were upregulated and 12 of them were downregulated.5.Overexpression of miR-34 c inhibited porcine intramuscular adipocyte differentiation.miR-34 c is conserved in various species and the candidate targets were predicted by bioinformatic analysis.These targets were involved in AMPK,mTOR,PI3K-Akt,FoxO1,insulin signaling pathway and so on.Of them,multiple pathways is closely linked to adipogenesis.Interestingly,overexpression of miR-34 c suppressed the intramuscular adipocyte adipogenesis and decreased the mRNA and protein levels of PPAR?,FABP4 and FASN.6.The dual luciferase reporter assay indicate E2F3 and KLF4 are the targets of miR-34 c.7.Overexpression of miR-34 c suppressed the cell cycle protein Cyclin B expression.In addition,flow cytometer analysis showed that overexpression of miR-34 c increased the population of G2 phase cells(P < 0.05)and decreased that of S phase cell(P < 0.01).Consistently,CCK-8 activity significantly decreased in overexpressed cell(P < 0.05).In summary,in this study we compared the difference of cultured intramuscular fat cell from two depots of skeletal muscles.the miRNAs profiles were systemically identified from these in vitro cultured cells during differentiation.Importantly,we reveal the biological function of miR-34 c in intramuscular adipocyte proliferation and adipogenesis.These findings may provide reference for elucidating the mechanism of porcine intramuscular fat deposition.