Identification Of MicroRNA Regulatory Elements In Porcine Skeletal Muscle And Adipose Tissue And Mechanism Of Myoblasts Adipogenesis

Muscle quality is a long-term concern in pig breeding.Intramuscular fat is a key factor affecting pork quality and is highly correlated with pork flavor.On the other hand,ectopic fat production could lead to a variety of human muscle metabolic diseases.It shows that skeletal muscle and adipose tissue are independent and closely related.Previous studies have shown that skeletal muscle and adipose tissue have crosstalk,and myoblasts can be trans-differentiated into adipocytes.However,the specific mechanisms by which miRNAs play roles in this process,such as regulatory elements that control their transcriptional activity,transcription factors,etc.,are still unclear.Therefore,this study applied Chromatin immunoprecipitation followed by sequencing(ChIP-Seq)and Assay for Transposase-Accessible Chromatin with high throughput sequencing(ATAC-Seq)techniques to comprehensively analyze the regulatory elements of miRNA and its regulation in porcine skeletal muscle and adipose tissue.We used myoblasts as a model to further analyze the regulation mechanism of miRNA in the process of adipogenesis.The main results of this study are as follows:(1)Established the experimental platform for ChIP-Seq and ATAC-Seq techniques of tissue and cell in this laboratory:We explored and optimized a series of experimental processes in porcine skeletal muscle and adipose tissue,such as tissue lysis condition,chromatin sonication,co-immunoprecipitation,Tn5 transposition,and library construction.The strict quality control of the experimental processes and data quality was applied.The data obtained in this study is consistent with the data quality standards required by the Encyclopedia of DNA Elements(ENCODE)project.(2)Identification of promoter,enhancer elements,and open chromatin regions in porcine skeletal muscle and adipose tissue on the whole genome:According to the ChIP-Seq enrichment of the histone factors H3K4me3 and H3K27ac,a total of 22670 and 30045 enhancers,17735 and 20438 promoters were identified in porcine skeletal muscle and adipose tissue,respectively.Furthermore,the number of active promoters accounted for the 90%of the total promoters.According to the ATAC-Seq results,approximately 57936 and 66785 open chromatin regions were identified in porcine skeletal muscle and adipose tissue,respectively.(3)Common and specific promoter signals,enhancer signals,and open chromatin regions identification in porcine skeletal muscle and adipose tissue:The signals mainly divided into three categories:acquired signals in skeletal muscle tissue(Gain),shared signals in both tissues(Stable),and lossed signals in skeletal muscle tissue,that is to say,the acquired signals in the adipose tissue(Loss).The GO function enrichment analysis was performed on the promoter and enhancer signals.The Gain regions were significantly enriched with signaling pathways related to muscle organ development.The Loss regions were significantly enriched by adiponectin and lipoprotein-mediated signaling pathways.Motif analysis of open chromatin regions:Gain regions were related to MEFs,MYOG,MYF5,and other transcription factors regulating skeletal muscle development.Loss regions were enriched in CEBPB,ATFs,PPARG,and other transcription factors regulating adipogenesis.These promoter and enhancer signals and open chromatin regions were shown to be tissue specific.(4)The miRNAs promoter and enhancer analysis of porcine skeletal muscle and adipose tissue:there were obvious signal distributions of H3K4me3,H3K27ac and ATAC-Seq in the range of 50 kb near the miRNA in both tissues.In porcine skeletal muscle and adipose tissue,a total of 341 miRNA promoters were identified,among them,207 were active promoters.63.02%of the promoters predicted by this study have CpG island enrichment.Because CpG island mainly located in the gene promoter region,the promoter identified in this study was reasonable and reliable.In addition,373 miRNA potential enhancers were identified.Approximately 50%of the miRNA promoter and enhancer signals were located within 20 kb of the miRNA.(5)In porcine skeletal muscle and adipose tissue,the miRNA promoter and enhancer regions overlaped with the open chromatin region:86.57%of the miRNA promoter region and 60.96%of the miRNA enhancer region overlaprd with the ATAC-Seq signal,indicating that the ATAC-Seq signal could well cover the predicted regions of H3K4me3 and H3K27ac.The motif enrichment analysis of these regions indicated that the expression of miRNA is regulated by transcription factors.(6)Myoblasts,as research model,can induce adipogenesis in adipogenic differentiation medium,and the open chromatin regions change:when the myoblasts reach 80%density in the growth medium,the adipogenic differentiation medium is induced.Induction of 7d cells resulted in the formation of a large number of lipid droplets accompanied by up-regulated expression of the key adipogenic genes,such as Cebpa,Fabp4,AdipoQ,Dio2 and Cidea.There is a significant difference in the open chromatin region of ATAC-Seq data between the proliferative cells and the induced adipogenic cells.(7)The analysis of differentially expressed(DE)miRNA in porcine skeletal muscle and adipose tissue and DE miRNA during myoblast adipogenesis:In this study,a total of 160 DE miRNAs were identified in porcine skeletal muscle and adipose tissue,and 52 DE miRNAs during myoblast adipogenesis,of which 21 DE miRNAs were also enriched in pigs.GSEA enrichment results showed that more than 70%of DE miRNAs were involved in the regulation of gene expression during the adipogenic process.The target genes of these DE miRNAs were mainly enriched in Wnt,MAPK and Insulin signaling pathways.(8)The open chromatin regions in the range of 20 kb from DE miRNA in tissues and cells were mainly enriched in AP-1 related transcription factors and KLFs family:Transcription factors AP-1 and KLFs regulated DE miRNAs,DE miRNAs acted on their target genes,which in turn affected signaling pathways such as Wnt,MAPK,and Insulin and finally regulated transcription axis Foxo1-Ppargc1a-Ppara-Rxrg-Pparg,which was involved in the trans-differentiation of myoblasts to adipocytes.These transcription factors played an important role in the regulation of DE miRNAs in skeletal muscle and adipose tissue,as well as adipogenesis process.