Identification Of MicroRNA Regulatory Elements And Study Of Myogenesis Mechanism Regulated By MicroRNA

Skeletal muscle is the tissue with the highest proportion of mammalian body weight,and its size directly affects the economic benefits of the pig industry.Therefore,the research on the development of pig skeletal muscle is particularly important.Many studies have shown that mi RNAs are closely related to the growth and development of the skeletal muscle.However,the specific mechanisms that mi RNAs play in this process,such as the regulatory elements that control their own transcription and the key pathways that regulate the growth and development of skeletal muscle,remain to be explored.In this study,C2C12 myoblasts were used as the model,and epigenetic strategies such as ATAC-seq and Ch IP-seq were used to comprehensively analyze the regulation mechanism of mi RNAs during C2C12 myogenic differentiation.Furthermore,the key molecular pathways of mi RNAs in regulating myogenic differentiation were explored through transcriptome analysis strategies and a series of functional verification assays.In addition,the expression features of mi RNAs in pig skeletal muscles at different developmental stages were preliminarily mapped.The main results of this study are as follows:(1)In this study,the model of C2C12 cell induced myogenesis was established.A total of 28 differentially expressed(DE)mi RNAs were identified on days 0,1,2,and 4 during cell differentiation,including 9 novel mi RNAs.Heat map clustering analysis showed that 13 mi RNAs were significantly down-regulated overall,whereas 15 mi RNAs clustered into another group that was upregulated overall.(2)ATAC-seq data were generated for C2C12 cells on day 0 and day 2.5 during cell differentiation.The fold change patterns of 113 normalized ATAC-seq intensities in the genomic region ±10 kb around the pri-mi RNA loci of DE mi RNAs were analyzed,and found that a significantly strong signal of ATAC-seq in the group of upregulated DE mi RNAs and a significantly weak signal of ATAC-seq in the group of downregulated DE mi RNAs were observed.These results confirmed that these DE mi RNAs were regulated by the states of chromatin structure.(3)Combined with the published Ch IP-seq data,the study found that 44.25% of the 113 ATAC-seq peaks around the mi RNA sequences were occupied by Myo D or Myo G,and the remaining regions were significantly enriched with AP-1 family,Ctcf and Bach2.Moreover,these transcription factors were expressed and their expression levels changed significantly during myogenic differentiation.These results suggested that these transcription factors bound in the open chromatin region might be involved in the transcriptional regulation of mi RNAs.(4)Further research found that the changes in Myo D intensities in the genomic region ±10 kb around the pri-mi RNA loci of DE mi RNAs were consistent with the differential expression pattern of mi RNAs.while Myo G expressed during the differentiation phase had high intensities around all DE mi RNAs.Furthermore,the majority of the Myo D and Myo G binding sites around these mi RNAs were colocalized in myotubes.The above results indicated that in the open chromatin region,the functions of Myo D and Myo G in regulating differential mi RNA expression during myoblast differentiation were reciprocally dependent.(5)A total of 4485 target genes of DE mi RNAs were obtained.These target genes were mainly enriched in muscle growth and development-related pathways,especially Hippo and MAPK signaling pathways.And heat map clustering analysis found that the expression of target genes in these two pathways indeed changed with cell differentiation,indicating that multiple mi RNAs might coordinate the regulation of myogenic differentiation through the two pathways.(6)This study generated the potential regulatory network of Hippo signaling pathway and 13 DE mi RNAs involved.The dual-luciferase reporter system showed that members of Hippo signaling pathway were indeed targeted by these mi RNAs.Furthermore,the function loss models of Hippo signaling pathway were constructed,and confirmed that Hippo signaling pathway were indeed involved in the regulation of C2C12 myoblast differentiation and played different functional roles by Q-PCR,Western blotting and immunofluorescence experiments,specifically,Yap1 inhibited but Wwtr1 promoted the differentiation process.(7)The potential regulatory network of 11 DE mi RNAs that co-participated in MAPK/Erk signaling pathway to regulate myogenic differentiation was also generated.The verification results of the dual-luciferase reporter system also indicated that members of the signaling pathway were indeed targeted by these DE mi RNAs.Combined with RNA-seq data analysis,the MAPK/Erk pathway might play a negative regulatory role in the process of myogenic differentiation.(8)Combined with two published mi RNA-seq datasets of different development stages of porcine skeletal muscle,it is found that a variety of high expressed mi RNAs existed in different developmental stages of skeletal muscle,and there were more abundant types of high expressed mi RNAs during the embryonic stage,of which 20 DE mi RNAs existed in the entire skeletal muscle development process.These results indicated that multiple mi RNAs might form a combined effect to regulate the growth and development of pig skeletal muscle,had a more critical molecular effect during the embryonic development of skeletal muscle in pigs.