Skeletal muscle growth and development are a complex process, which directly determine the meat production and quality in the pig industry. Skeletal muscle is mainly composed of muscle fibers, basement membrane, muscle satellite cells and nerves. Study found that the numbers of muscle fiber have been fixed before the pigs were born, indicating that muscle fiber development is mainly determined during the embryonic period30,31. Muscle fiber development takes place in two waves in pig embryonic, the first wave of muscle fiber formation occurs from 30 to 60 days and the second wave occurs from 45 to 90 days32,33. In our study, we investigated lincRNAs expression profile in days 33, 65, and 90, which included the primary, second, and final waves of muscle fiber development33. Even though the previous studies have showed lincRNAs associated with muscle growth in pig, the dynamic process of expression profile of lincRNAs in embryonic muscle fibers is rare, and our study provides theoretical basis for new exploration in the future.
Based on RNA-seq data published in NCBI, we compared whole gene expression profile in muscle tissue from druoc in differentially development periods. Through a series of transcriptome pipeline analysis, there were 229 putative lincRNAs identified using RNA-seq sequencing, we predicted 39 novel lincRNAs that were not annotated from the nine muscle libraries, which enrich the pig lincRNA annotation and the specific features need to be further investigated in the future. Moreover, we performed a characteristic analysis of putative lincRNAs, involving in transcript and exon length, exon numbers and FPKM, the results showed that the similar characteristic of shorter transcript length, longer exon length, fewer exons, and lower expression levels compared with previous reports34,35. Meanwhile, the reliability of the analysis is further improved.
We identified 95 DE lincRNA genes and 2638 DE protein-coding genes based on a designed pipeline. Previous studies have shown that there were a large number of lncRNA located within known QTL regions36. To understand the relation between DE lincRNAs and QTL, we performed also QTL localization analysis for differentially expressed lincRNAs. Some QTLs are involved in large regions,so that multiple genes are located on the same QTL, or multiple QTLs have the same gene location. In among, the specific mechanism may need to be verified by subsequent experiments.
To explore the potential function of DE lincRNAs, we investigated the regulation of lincRNAs on gene expression through cis and trans regulation26. For the cis-regulation of DE lincRNAs, we found these genes have been shown to be associated with muscle cell proliferation and fat deposition. For example, DLK1 was a critical factor in regulating skeletal muscle development and regeneration through Notch dependent37. Previous studies found that PPARA was involved in the regulation of fat deposition in porcine subcutaneous fat and longissimus dorsi muscle38,39. Besides, myofibrillar structural protein myomesin-3 (MYOM3) was not only associated with muscular dystrophy related proteins and muscle strength, which could be a potential biomarker for monitoring of muscular dystrophy, but was hymethylated in ischemic cardiomyopathy40,41. Among them, DEL-MSTRG. 31882 and its potential target gene with patatin like phospholipase domain 4 (PNPLA4) showed significant positive correlation at the expression level. Therefore, we inferred that DE lincRNAs RNA modulates differences at different developmental stages by regulating their potential target genes.
In this study, we investigated Gene Ontology and KEGG pathways analysis of potential target genes of DE lincRNAs, and found that skeletal muscle organ and tissue development processes, muscle contraction, striated muscle cell development were some of significantly enriched GO terms. This results suggest that identified DE lincRNAs have important impact in the skeletal muscle. Regulation of glucose import, regulation of glucose transport, and insulin receptor signaling pathway also significantly enriched, which were important ways of obtaining and transporting energy, we deduce that DE lincRNAs could participate in the regulatory mechanism of skeletal muscle development through mediating cellular energy responses. Our KEGG pathway analysis showed that significantly enriched pathways including MAPK signaling pathway, TGF-beta signaling pathway, Wnt signaling pathway, ECM − receptor interaction, regulation of kinase activity. Previous studies have confirmed that TGF-beta signaling pathway contributes to muscle development in mice42. Moreover, the extracellular matrix (ECM) is a network of structures surrounding muscle fibers, providing a close connection with cell proliferation, differentiation and metabolism. Therefore, we infer that DE lincRNAs could contribute to the differences in skeletal muscle development. In addition, some cardiac diseases, such as viral myocarditis, dilated cardiomyopathy, and hypertrophic cardiomyopathy, were also significantly enriched, these results suggest that DE lincRNAs may have an important effect on myocardial development.