| Folate deficiency during pregnancy can cause fetal intrauterine growth restriction, of whichthe skeletal dysplasia is a major manifestation. However, the effect of folate deficiency onskeletal muscle development and its molecular mechanisms are unknown. Factors influencingmuscle development are very important in the formation of porcine meat quality trait. Thisresearch was to discuss the effects of folate deficiency during pregnancy on development ofthe skeletal muscle in piglets, aimed at providing strong experimental support for meat qualityimprovement and clinical folate nutrition. Firstly, we take the pig as the animal model todetermine the effect of maternal folate deficiency on the skeletal muscle transcriptome ofpiglets. In our study full-sibling Landrace (LR) and full-sibling Chinese local breed Laiwu(LW) pigs were used for reciprocal cross matings, and sows were fed either a folate deficientor a normal diet during early-mid gestation. Longissimus dorsi (LD) muscle samples werecollected from newborn piglets and a4×44K Agilent porcine oligo microarray was used fortranscriptome analysis of porcine LD muscle. The results showed that folate deficiency duringearly-mid pregnancy affected piglet body weight, LD muscle fiber number and content ofintramuscular triglyceride. The microarray results indicated that3154genes weredifferentially expressed between folate deficient and normal piglets from the LR♂×LW♀cross, and3885differentially expressed genes (DEGs) in the ones from the LW♂×LR♀cross. Most of the genes that are regulated by folate deficiency in the LD muscle of pigletswere different between LR♂×LW♀and LW♂×LR♀crosses. From functional analyses,sow folate deficiency affected almost all biological processes in the progeny. Lipidmetabolism-related genes and associated metabolic pathways were regulated extensively byfolate deficiency, especially in LR♂×LW♀cross piglets.On the other hand, mouse C2C12myoblast cells are an system widely used to study genesthat regulate muscle growth and differentiation in vitro. In this study, we take C2C12as thecell model to study the effect of folate deficiency and supplement on proliferation anddifferentiation of myoblasts and its possible mechanisms. C2C12cultured in D2429(mediumlack of folic acid) was used to analyze the effect of folic acid on myogenesis. According to thefolic acid concentration, three groups were divided: folate deficiency (Folate-0mg/l, L),normal folate (Folate-4mg/l, N) and high folate supplement (Folate-40mg/l, H).We firstly studied the effect of folate on proliferation and differentiation of C2C12cells, and found thatfolate deficiency can promote proliferation and inhibit the differentiation of C2C12cells,while high folate can promote the proliferation and differentiation of C2C12cells. In theprocess of C2C12proliferation, the mRNA expression of MyoD in normal folate medium wassignificantly higher than that in folate deficiency and high folate medium (P<0.05). Bybisulphite sequencing, we found that five CpG sites located from-16bp to+26bp DNAaround TSS of MyoD were hypermethylated in high folate group, while were hypomethylatedin normal folate. We also found that in the process of C2C12differentiation, the expression ofMyogenin was enhanced along with the increase of folic acid concentration, and that the2nd,3rd and4th CpG loci located from-374bp to+6bp DNA of Myogenin TSS werehypermethylated in normal folate medium, the7th and8th CpG loci were hypermethylated inhigh folate and the5th and9th ones were hypermethylated in folate deficiency group.However, DNA methylation of the first exon of Myogenin was not significantly differentamong three groups.In addition, we tested the influence of the folic acid on genome-wide methylation changesduring C212differentiation by MeDIP-chip. We found that hundreds of genes werehypermethylated or hypomethylated in each group, and differentially methylated regions(DMRs) were found between every two groups both in promoter and in CpG islands. Forexample,180DMRs (84hypermethylated and96hypomethylated) exist in promoter betweenN and L,199(95hypermethylated and104hypomethylated) in H vs L and201(104hypermethylated and97hypomethylated) in H vs N. In methylation profile, we found onlyfew common DMRs between N vs L and H vs N. It showed that different genes wereregulated in folate deficiency and high folate group to regulate C2C12differentiation.Correlation analysis between methylation profile and expression profile revealed that somegenes were regulated by methylation status directly, but at the same time, DNA methylationwas not the only factor to regulate gene expression.In conclusion, our results showed that folate deficiency in sows during early-midpregnancy altered the transcriptome of the longissimus dorsi muscle in the offsprings, thusaffecting the process of myogenesis in fetal pigs. We also found that folate deficiency andsupplement can influence the proliferation and differentiation of C2C12, and that DNAmethylation is an important factor regulating genes underlying myogenesis. This studyprovides genetic and epigenetic basis for meat quality improvement and folic acid nutrition. |
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