Controlling factors that contribute to average daily weight gain is crucial for the Hanwoo beef industry, as a high occurrence of low weight can result in significant economic losses. With the escalating production costs due to global feed price increases, reducing the age of slaughter has been acknowledged as a viable method to enhance farm income. Thus, it is imperative to explore strategies that allow for a shorter age of slaughter while simultaneously ensuring optimal daily weight gain and meat quality. Consequently, numerous endeavors have been undertaken to enhance meat quantity by regulating the influential factors including dietary treatment, breed type, age, environmental and genetic factors related to carcass weight 11,12. Therefore, a comprehensive understanding of the genetic architecture of key growth and livestock traits is essential for identifying new genes and genetic pathways that can be leveraged through genomic selection to improve economic traits in livestock breeding programs.
In South Korea, the psoas major muscle holds significant importance due to the high demand for steak. The psoas major, also known as filet mignon, is a highly prized cut of beef known for its exceptional tenderness and mild flavor. It is a lean muscle located along the spine of the animal and is considered one of the most tender and desirable cuts. Similarly, the rump semimembranosus muscle holds economic value in the Hanwoo beef industry. Producers and sellers often prioritize this cut to cater to consumer preferences and meet market demand due to less adipose accumulation and lean meat, thereby contributing to the overall profitability of the industry 13.
Hence, the current study was carried out transcriptome analysis on these three LD, SB, and PM skeletal muscles to provide the novel molecular functions of the tissues, which may influence the average daily body weight gain in Hanwoo steer. In our study, we selected male Hanwoo cattle with a significance difference in ADG group (high ADG ≥ 0.88 kg and low ADG ≤ 0.78 kg). The result of this study revealed that DPP6 (LD), IL22RA1 (SB), and U6 (PM) genes were the top upregulated genes. Among these genes, DPP6, also known as dipeptidyl peptidase-like protein 6, is a gene that encodes a protein involved in neuronal signaling and ion channel regulation 14. It has been suggested as candidate associated with altered lipid profile 15, muscle atrophy in human 14, and the rib eye Muscle Area in Hu Sheep 14. According to Zhao et al., it is plausible that DPP6 may have some influence on muscle physiology and potentially contribute to muscle wasting or atrophy 14. IL22RA1 (interleukin 22 receptor subunit alpha 1) has been shown to be receptor for interleukin-22 (IL-22), a cytokine involved in various biological processes, including cell proliferation and tissue regeneration in animals and human 16. Therefore, DPP6 gene can be suggested as one of good candidate gene for ADG variation in Hanwoo. While IL22RA1’s role in muscle growth is not extensively studied, there is evidence suggesting its secondary involvement in muscle physiology 16–18. IL22RA1 being identified as a candidate gene associated with health, adaptation and reproduction traits in cattle 19. For PM tissue, small nuclear RNAs (U6) plays catalytic role at the core of the spliceosome 20 but straight function associated to muscle development or growth performance in cattle not been reported before. Furthermore, a number of genes that were downregulated involved in diverse function linked to cell death, metabolism, cell growth and development were screened as possible candidate genes for ADG trait. Of the top downregulated DEGs, myosin light chain 6B (MYL6B), fibroblast growth factor 6 (FGF6), acyl-CoA synthetase bubblegum family member 1(ACSBG1), two pore channel 3(TPC3), polymeric immunoglobulin receptor(PIGR), Wnt family member 16 (WNT16), alpha-2-glycoprotein 1, zinc-binding (AZGP1), and forkhead box Q1(FOXQ1) genes were found to be involve in various function associated to growth, development and metabolism.
The total upregulated and downregulated significant DEGs were used to determine the GO and KEGG pathway analysis, which was then used to identify significant enriched pathways for the three tissues. Based on the pathway analysis, JAK-STAT signaling pathway was detected as the most significant enriched pathway in LD tissue. In muscle development and regeneration, this pathway plays a vital role in regulation of adipogenesis, process of fat cell development, controlling myoblast proliferation, differentiation, and the formation of muscle fibers 21–23. Cytokines and growth factors like IL-6, LIF, and IGF-1 activate the JAK-STAT pathway to facilitate muscle cell growth and promote myogenic differentiation 24. A previous GWAS study identified candidate genes associated with the body weight trait in Chinese sheep, revealing their involvement in the JAK-STAT pathway 25.
Among the upregulated DEGs in JAK-STAT pathway, cyclin dependent kinase inhibitor 1A (CDKN1A) could be an important target. We observed a significant increase in the expression of the CDKN1A gene, which is associated with the regulation of the G1/S checkpoint and is known to be crucial in cellular senescence during preimplantation embryo development in humans 26. Zhang et al. reported that CDKN1A regulates myogenesis process in muscle stem cells 27. Recently, CDKN1A gene has been reported as strong candidate gene for high fat milk production trait in dairy cattle (Chinese Holstein population) 28–30. Also, the CDKN1A gene demonstrated common enrichment in conjunction with other pathways. Therefore, based on previous CDKN1A results, we speculate that upregulation of CDKN1A expression in high ADG group is closely related to myogenesis or lactation process. Moreover, another vital gene, SOCS3 (suppressor of cytokine signaling family 3) known as negative regulator of JAK-STAT, was detected to be downregulated in our study 31. It indicates that the downregulation of SOCS3 could leads to STAT3 phosphorylation which further may promotes lipid metabolism, cell cycle progression, differentiation 32,33. Although future studies are expected to provide more comprehensive insights into the specific roles of both the genes in JAK-STAT pathway and other regulatory mechanisms in muscle growth, ultimately leading to weight gain. The findings indicate that the JAK-STAT pathway, along with other pathways, may affect the ADG trait in Hanwoo through its influence on myogenesis, lactation, cell growth and differentiation.
In SB tissue, the prolactin signaling pathway emerged as the most significant pathway. The prolactin signaling pathway has significant involvement in the regulation of metabolism, immune system function, milk synthesis, cell growth, survival and pancreatic development. Activation of this signaling pathway was reported to promote cell growth and survival 34. Prolactin hormone belongs to this pathway has been reported to be involve in smooth muscle cell proliferation function in mammals through protein kinase C-dependent mechanism 34,35. The prolactin signal pathway activates after the interaction of prolactin with its specific membrane receptor, which further exerts its effects through the activation of specific signaling pathways, including the JAK-STAT and MAPK pathway 34. According to Chu et al., the prolactin receptor (PRLR) is a candidate gene for prolificacy of small tail han sheep 36. In Egyptian Buffaloes, the Prolactin receptor (PRLR) has shown promise as a genetic marker for evaluating milk production and quality traits 37. The current experiment revealed a significant upregulation of the PRLR gene by a log2FC of 3.05. This possibly suggests that the ADG trait is likely to be influenced by the stimulation of prolactin-signaling pathway which leads to activation of cascade of protein kinase C-dependent mechanism, and JAK-STAT pathway. In this study, another gene associated with the prolactin signaling pathway, MAPK13, exhibited downregulation. MAPK13 encodes a protein belonging to the mitogen-activated protein (MAP) kinase family, which serves as a convergence point for various biochemical signals 38. MAP kinases are involved in numerous cellular processes including proliferation, differentiation, transcription regulation, and development 38,39.
Subsequently, the KEGG analysis identified PPAR signaling pathway as a potential candidate pathway that was the most significant pathway found from the transcriptome analysis of PM tissue. The PPAR signaling pathway has been examined in many studies with regard to its function in adipose tissue influences adipogenesis, glucose metabolism, and adipokine secretion in cattle 8,40. Recently, He et al., described that 13 tag single nucleotide polymorphisms in the PPAR signaling pathway are associated with porcine meat quality traits 41. A study on Hanwoo cattle investigated the gene expression patterns associated to PPAR signaling pathway. The results of this study demonstrated a significant association between PPAR signaling and genes involved in fatty acid oxidation. This association ultimately led to an increase in triglyceride formation through ATP production 8. In Hanwoo cattle, the finishing period in cattle starts at 13–15 months of age and the adipose tissue starts to accumulate in muscle during 13 to 30 months of age 42,43. Hence, it can be hypothesized that the PPAR signaling pathway plays a role in controlling adipogenesis and influencing the capacity of adipose tissue to store lipids, thereby affecting the fat mass in Hanwoo muscles. Consequently, by regulating the size and quantity of adipocytes within the muscle, this pathway may impact the average daily gain characteristic in Hanwoo cattle.
Activation of PPARs (peroxisome proliferator-activated receptors) within this pathway induces several downstream biological pathways, including those related to lipid metabolism, adipocyte differentiation, and gluconeogenesis in liver and skeletal muscle 44. In current study, we identified the major downstream pathways associated with PPARs signaling pathway such as adipocytokine signalling pathway and regulation of lipolysis in adipocytes. A muscle transcriptome study conducted earlier identified the adipocytokine signaling pathway and PPAR signaling pathway as potential candidate pathways associated with the longissimus thoracis muscles in meat-type sheep from India 45. The genes involved in PPAR signaling pathway were ADIPOQ (Adiponectin, C1Q and collagen domain containing), PCK2 (Phosphoenolpyruvate carboxykinase 1), FABP4 (Fatty acid binding protein 4, adipocyte), PLIN1 (perilipin). FABP4 gene reported to be facilitates the uptake of fatty acids and their intracellular trafficking, contributing to the process of adipocyte maturation and lipid accumulation 8,46. ADIPOQ is a well-known homeostatic factor for regulating glucose levels, and lipid metabolism47 and reported to be candidate gene for carcass trait. PLIN1 plays a crucial role in controlling the levels of triglycerides and the size of lipid droplets in adipocytes, contributing to the maintenance of lipid balance48. It is also suggested as possible robust candidate gene marker for body weight in cattle breeding programs49. Furthermore, the SLC2A1 gene enriched in adipocytokine signaling pathway was upregulated by more than 2 fold here in PM tissue. SLC2A1, a glucose transporter, has also been implicated in facilitating the transport of fatty acids 50. In summary, it is postulated that the mentioned genes may facilitate the accumulation of intramuscular fat in cattle by controlling the synthesis, transport of fatty acids, lipid balance by regulating the respective signaling pathways and potentially affecting the ADG trait. However, a more comprehensive understanding of the precise regulatory mechanisms involved requires further exploration in future studies.
In the present study, GO analysis revealed that DEGs of LD tissues were mainly enriched in branching involved in labyrinthine layer morphogenesis, retinoic acid metabolic process, T cell chemotaxis and detoxification of copper ion biological process. For the SB tissue, we found significant biological process GO terms that are the sodium ion transmembrane transport and response to estrogen biological process. In PM tissue GO analysis, the result revealed some important biological process GO terms that are interleukin production, fat cell differentiation, carbohydrate metabolic process, ion transmembrane transport, insulin resistance and etc. Among all these GO, ion transmembrane transport facilitates nutrient absorption, carbohydrate metabolism provides energy, and fat cell differentiation contributes to energy storage, all of which are essential processes for weight gain in cattle. Understanding and managing these processes can help optimize weight gain and improve cattle breeding and production practices. These biological processes have been reported to be have dominance effect on porcine and cattle final weight and back fat thickness 19,51,52. Moreover, the GO network analysis revealed that several essential genes, which were commonly shared among significant GO terms, exhibited significant upregulation. It indicates their potential functional importance in the analyzed biological system. For example, FZD5 involved in two different GO terms are found to be upregulated in our datasets. These genes likely play crucial roles in the biological processes, cellular components, or molecular functions represented by the enriched GO terms. Further investigation and validation of these genes can provide insights into their specific roles and mechanisms within the biological system under study. It would be beneficial to conduct additional experiments or analyses to confirm their functional significance and elucidate how their upregulation contributes to the observed biological phenomena like the ADG trait variation in Hanwoo cattle.
Furthermore, we constructed a protein-protein interaction (PPI) network to gather valuable interaction information regarding the differentially expressed genes (DEGs). By aligning the PPI networks, we can delve deeper into the fundamental relationships among the differentially expressed genes (DEGs) by associating them with genes and protein complexes within the network pathways 53. The PPI network in the current study was created with total significant DEGs (P-value of ≤ 0.05, log2FC of ≥ 1.5) collected from three experimental tissues. By integrating the DEGs from these tissues, the PPI network was generated to capture correlation patterns of gene expression across samples, the new interactions and relationships between proteins that tend to be co-regulated or functionally related 54. In present investigation, we identified 12 hub genes that are JUN (transcription factor Jun), CDKN1A (cyclin dependent kinase inhibitor 1A), CDH1 (cadherin-1), TP53 (tumor Protein P53), FZD5 (frizzled class receptor 5), PPARG (Peroxisome proliferator-activated receptor gamma), LEP (leptin), IL4 (interleukin 4), PTGS2 (prostaglandin-endoperoxide synthase 2), AXIN2 (axin-related protein 2), CDH2 (cadherin-2) and MYC (MYC proto-oncogene). These findings highlight the centrality of these hub genes and suggest their involvement in critical biological processes or pathways associated with growth performance in Hanwoo.
Among all the hub genes, notably the CDKN1A and FZD5 were spotted to be upregulated (> 1 fold) in all the three experimental tissue samples. In our study, we have identified CDKN1A as a key hub gene that works as inhibitor in cell cycle and control myogenesis process in muscle stem cells 27. It has been described as candidate gene associated with carcass quality trait and milk production in beef cattle 55. As mentioned earlier in our pathway analysis study, we emphasized here the importance of this gene. Furthermore, our module analysis indicated that CDKN1A is associated with module 1. Interestingly, in our current experiment, we observed that CDKN1A is enriched in various pathways, including those related to different types of cancers, Hepatitis B, Epstein-Barr virus infection, PI3K-Akt signaling pathway, p53 signaling pathway, and JAK-STAT signaling. These findings collectively suggest that CDKN1A may influence muscle growth in Hanwoo cattle by regulating the muscle differentiation at the myogenin step, thereby subsequently affecting the ADG trait. However, more research is needed to confirm this theory. The other gene, FZD5 is a protein coding gene, which functions as transmembrane receptor and mediates Wnt ligands binding in Wnt signaling pathway and plays role in the control of tissue regeneration, cell proliferation and cell differentiation 56,57. One recent study has proposed that the activation of FZD5 by Wnt protein could potentially play a role in regulating adipose differentiation 56. Therefore, it could be valuable to study further on FZD5 functions in the muscle growth and ADG trait.
In current study, the top three modules were selected for further investigations of PPI network. Our result presented that the genes in all the modules are involved in several important signaling pathways including PI3K-Akt signaling pathway, Wnt signaling pathway, JAK-STAT signaling pathway, PPAR signaling pathway, IL-17 signaling pathway, and C-type lectin receptor signaling pathway. PI3K-Akt signaling and Wnt signaling pathway were found to be common in two modules i.e. module 1 and module 3. A considerable number of studies have reported that the PI3K-Akt signaling and Wnt signaling pathways are the key pathways for regulating cell propagation, adipose proliferation, fat deposition, and muscle development in bovine thus subsequently effect the meat production and carcass traits in cattle 58–61. Notably, the outcomes from GO/KEGG analysis were matched to the functional annotations of genes in the most top three modules of PPI network. Additionally, it is noteworthy to mention that genes enriched in module 1 (CDKN1A, AXIN2, CDH2, MYC, and TP53), module 2 (JUN, PPARG, PTGS2), and module 3 (TP53, FZD5, IL4, and MYC) were found as hub genes as well. The speculation that the identified genes, which are differentially regulated and associated with various pathways in different tissues, could be potential candidate genes affecting the ADG trait is reasonable. However, it is crucial to note that further intensive investigations are necessary to validate and confirm the significance of the identified candidate genes and pathways.
overall, based on the findings from the analysis of DEGs, KEGG pathway enrichment, PPI-hub gene, modules, and insights from relevant literature, we have identified three genes (DPP6, CDKN1A, and FZD5) that show promise as potential candidate genes influencing ADG trait in Hanwoo steer.