In this study, we first examined the apparent differences in muscle fibres of different breeds of beef cattle. The results showed that there were obvious differences in the apparent observation of the muscle fibres of HE stained paraffin sections of the longissimus dorsi of Black cattle and Luxi cattle. The length of a single muscle fibre of Black cattle was significantly longer than that of Luxi cattle, and the number of nuclei in each muscle fibre was also greater. The border between the muscle fibres of Black cattle was clearer and rounder than that of Luxi cattle. IPP software analysis showed that there were significant differences in the diameter and length of muscle fibres (P < 0.05) but no significant differences in the number, density or area of other muscle fibres (P > 0.05). The occurrence of these differences may be the key factors leading to the differences in meat production performance and meat quality of the two breeds of cattle after birth, which was also the research basis of this study to explore the underlying molecular regulatory mechanism.
We used RNA-seq technology to study the expression of circRNA in the longissimus dorsi muscle of different breeds of beef cattle. A total of 14640 circRNAs and 4201 parental genes were detected. circ0013465 (UBE2D1), circ0011592 (UBE3A) and circ0001880 (MYL1) were the most highly expressed in the two libraries. PROSITE-ProRule annotation of these proteins (UBE2D1 and UBE3A) showed that they are involved in protein ubiquitination, which is part of the protein modifications that regulate cell metabolism within eukaryotes [10]. MYL1 is a crucial protein for adequate skeletal muscle function and belongs to the myosin family [11]. The ubiquitin proteasome system (UPS) is mainly responsible for the increased protein breakdown observed in muscle wasting. The Ube family of E3 ligases is a class of enzymes (i.e., troponin I, myosin heavy chains and actin) that can guide the degradation of major contractile proteins. Their catalytic activity depends on the covalent binding of polyubiquitin chains catalysed by a specific E2 on the substrate [12]. Studies have shown that UPS can control almost any muscle mass and recovery process in catabolism. The muscle-specific E3 ligase UBE family participates in the targeting of actin, myosin, troponin and other major contractile proteins [13], indicating that the high expression of circRNAs plays a certain role in muscle development and redifferentiation.
According to the fold change > 1.5 and P < 0.05 criteria, 655 differentially expressed circRNAs were identified, corresponding to 467 parental genes, 267 of which were upregulated and 388 downregulated in Luxi cattle. The function of a circRNA is reflected in its parental gene. Because there is no information about the annotation of circRNAs at present, we annotated the parental genes of differentially expressed circRNAs. As a result, is the genes were annotated in 65 different GO terms, which mainly play a role in biological processes such as regulation of cell process, regulation of metabolic process and part in the cell. We identified 29 related terms. There were 60 different genes associated with muscle growth and development. The genes with higher enrichment were gene 1570 (TTN), gene 23041 (MYBPC2), gene 6914(MYBPC1), gene 1832 (NEB), and gene 22584 (MYH15). All of the above genes participate in muscle growth and development, and their corresponding circRNAs also play a role in this process. The number of circRNA parental genes in the different samples was significantly different. The difference reflects the cumulative effect of circRNAs on expression characteristics. Based on the KEGG pathway database, we further analysed the circRNAs and found that the AMPK signalling pathway, cellular signalling pathway, and cellular signalling and alternative signalling in cardiomyocytes were the most significantly enriched pathways. In our study, based on the above enrichment results, we identified 15 distinct enriched pathways related to muscle growth and development, including the AMPK signalling pathway, MAPK signalling pathway and adaptive signalling in cardiometrics, which influence muscle fibre processes [14,15]; the MTOR signalling pathway and Wnt signalling pathway, which are involved in the regulation of skeletal muscle development and regeneration [16 − 18]; the PPAR signalling pathway, which is involved in the regulation of intramuscular fat deposition [19] and the cytoskeletal signalling pathway. According to the statistical data, the genes with higher enrichment levels were gene 20196 (AKT3), gene 1084 (PIK3CB), gene 25426 (PIK3R1), gene 31865 (MAPK8), gene 21361 (MYL2), and gene 6914 (MYBPC1), which could indicate that the circRNAs produced by these genes may play a role in the growth and development of muscle through these pathways. Combining these results with the above results, we identified 7 parental genes (TNN, MYBPC1, NEB, MEF2C, MYH7, PPP2R3A and RYR1) and 38 corresponding circRNAs. Comparing these results with previous research results, significant differences were observed in the expression of circRNAs related to the muscle development of different breeds of cattle, suggesting that circRNAs may play an important role in muscle development. Whether these circRNAs have specific functions and what the functional mechanism is need to be studied further.
circRNAs can play important roles by regulating the transcription and expression of their parental genes [20]. At present, there is a relatively limited understanding of the details of the formation of circRNA and its functional mechanism. circRNA can be obtained by transcription of protein-coding genes or intergenic regions [21]. The formation of circRNA from a protein-coding gene is caused by the variable splicing of the parental gene [22]. Therefore, there should be a certain correlation between a circRNA and its parental gene expression. We found that one source gene may produce multiple circRNA subtypes. For example, the MYBPC1 gene can produce nine different circRNA subtypes. We obtained the FPKM value of the two varieties and found that both were differentially expressed. Although one source gene may produce multiple circRNA subtypes at the same time, only 3 or 4 of them have high expression levels, and the rest have low expression levels, which indicates that the cyclization of RNA in muscle is strictly regulated. To further understand the biological function and molecular function of the parental genes of significantly differentially expressed circRNAs, we predicted the interaction between circRNAs and miRNAs and constructed a network from the interaction data. The interaction network showed that a single miRNA may be correlated with multiple differentially expressed circRNAs, and there have been reports that circRNAs can competitively adsorb miRNAs [23]. Based on the high-throughput sequencing results, we selected 15 circRNAs related to muscle development as candidate circRNAs (circ0001048, circ0001103, circ0001159, circ0003719, circ0003794, circ0003721, circ0003720, circ0001519, circ0001530, circ0005060, circ0006589, circ0000181, circ0000190, circ0010558, circ0010577). In addition, the target miRNAs were predicted, the corresponding mRNA targets of the miRNAs were predicted, and the circRNA-miRNA-mRNA network was constructed to further study the regulation of muscle development. We will further verify this network in future experiments, which provides a new basis for the study of muscle development in cattle.
In addition to the above findings, there were pathways that were not found in our study that are known to be important and enriched for many parental genes and some that have been reported in previous studies to regulate muscle growth and development. The miRNAs that have been shown to be expressed specifically or preferentially in muscles are called muscle-specific miRNAs (muscle-specific microRNAs, myomiRs) [24] and include miR-1, miR-206, miR-128, miR-483, miR-2425-5p, miR-181a, miR-208a, miR-208b and miR-486 [25 − 27]. MiR-206 is specifically expressed only in skeletal muscle, and its targeted circRNAs (circ0001651, circ0010874, circ0010882, circ0010890, circ0010896, circ0012793) were not significantly differentially expressed. Whether these circRNAs have specific functions and what their functional mechanisms are need further study.