Integrated analysis of the whole-transcriptome of sheep skeletal muscle reveals the ceRNA regulation network related to muscle fiber formation in sheep

Background: The different muscle fiber types are closely related to meat quality. Tan sheep is famous for the fur breed of China, and the meat quality is among the best in various sheep breeds. The mutton of Tan sheep have many characteristics including tender, juicy, nutritious and unique flavor. In this study, the high-throughput RNA sequencing was used to identify the expression profiles of coding and non-coding RNAs from longissimus dorsi and biceps femoris samples obtained from Tan sheep, Dorper sheep and their hybrid Dutanhan sheep. Results: : The results of immunofluorescence staining showed that the proportion of slow-twitch fibers in longissimus dorsi tissue in Tan sheep (about 11.247%) was significantly higher than that in Dorper sheep (about 5.408%) ( P<0.05 ). A total of 819 difference expression (DE) lncRNAs, 354 DE circRNAs, 139 DE mRNAs, and 36 DE miRNAs were identified in high and low slow-twitch fiber ratio groups. Several key genes involved in signaling pathways that form different types of muscle fibers, including protein kinase AMP-activated catalytic subunit alpha 2 ( PRKAA2 ), EF-hand domain family member B ( EFHB ), folliculin interacting protein 2( FNIP2 ) and L-lactate dehydrogenase A chain ( LDHA ), had significantly different levels of expression in the two muscle tissues. Functional analysis suggested that the differentially expressed transcripts are involved in biological processes such as FoxO signaling pathways, AMPK signaling pathway and Ca 2+ signaling pathway. There were 26 lncRNAs, 115 circRNAs, 21 miRNAs and 4 genes formed 70 mRNA-miRNA, 31 lncRNA-miRNA, and 158 circRNA-miRNA interaction relationships, suggesting that complex regulatory networks of coding and non-coding genes underlie the muscle fiber formation in Tan sheep. The functional prediction results showed that many of these DE circRNAs, such as novel_circ_0017336, novel_circ_0007039, novel_circ_0017132, competing to bind with miR-23a, might act as competing endogenous RNAs (ceRNAs) to control PRKAA2, EFHB and FNIP2 level, respectively. RT-qPCR validation for randomly selected 4 DEGS ( PRKAA2 , EGR1 , MYL3 and FBXL5 ), 5 DEMs (miR-26a, miR-308-3p, miR-409-3p, miR-495-3p and miR-23a), and 4 DECs (circ_0017336, circ_0017132, circ_0017430, and circ_0007039) expression patterns were highly consistent with those obtained by RNA-Seq. Conclusion: Our study provides a foundation for understanding how lncRNA, circRNA, miRNA and genes interact to regulate muscle fiber formation in Tan sheep, Dorper sheep and their hybrid. We also constructed lncRNA/circRNA–miRNA–gene interaction networks to elucidate the complex molecular mechanisms underlying muscle fiber formation. The results extend our knowledge about the diversity of meat trait among different domestic animals and provide a foundation for studying novel mechanisms that control meat trait in Chinese indigenous sheep.