Skeletal muscle lncRNA profile associated with fatty acids in Nellore beef cattle Bruna Maria Salatta1, Maria Malane Magalhães Muniz2, Larissa Fernanda Simielli Fonseca1, Lucio Flavio Macedo Mota1, Caio de Souza Teixeira1, Gabriela Bonfá Frezarim1, Marta Serna-García1, Danielly Beraldo dos Santos Silva1, Angélica Simone Cravo Pereira3, Fernando Baldi1,3,4 & Lucia Galvão de Albuquerque1,4 This study aimed to identify differentially expressed (DE) long non-coding RNAs (lncRNAs) in muscle tissue of Nellore cattle clustered by their fatty acid profile. Longissimus thoracis muscle samples from 48 young bulls were used to quantify fatty acid (FA) (myristic, palmitic, stearic, oleic, linoleic, conjugated linoleic (CLA), α-linolenic and the groups of saturated fatty acids (SFA), monounsaturated (MUFA), polyunsaturated (PUFA), ω3, ω6, PUFA/SFA ratio and ω6/ω3) and to generate RNA- Sequencing data for transcriptomic analyses. The K-means analysis was used to classify the 48 animals into three clusters based on their FA patterns. The C1 had significantly (p ≤ 0.05) higher PUFA, ω3, ω6, linoleic and α-linolenic content. The proportion of SFA, myristic, palmitic and stearic were significantly (p ≤ 0.05) higher in C3, while C2 presented an intermediate profile. DE analyses were performed on three different comparisons, C1 vs. C2, C1 vs. C3 and C2 vs. C3, and 22, 28 and 22 DE lncRNAs (fold change > | 2 |, p-value < 0.01 and false discovery rate (FDR) < 0.05) were found, respectively. For three comparisons, the novel DE transcripts, lncRNA_15786.3, lncRNA_13894.1 and lincRNA_17393.3 interacted with CCN1, BNIP3, and CNOT2 genes, respectively, and appeared to contribute to a PUFA-enriched fatty acid profile. These genes are responsible for regulating the lipogenic genes, lipid metabolism, immune response and lipid synthesis. Meanwhile, the intergenic DE lncRNAs (lincRNA_18394.1, lincRNA_2526.3 and lincRNA_17681.1) were associated with the genes DDX1, EIF4E and APOL3, and appeared to contribute to a SFA-enriched fatty acid profile. The gene DDX1 was enriched by GO terms related to RNA splicing (GO:0008380), while the other genes (e.g., EIF4E and APOL3) were enriched to GO terms related to lipid transport (GO:0006869), localization (GO:0010876) and to cellular response to lipid (GO:0071396). These findings offer new insights into the biological mechanisms underlying the gene regulation of FA composition in beef and may provide a valuable foundation for further investigations regarding the interactions between lncRNAs and mRNAs, as well as their potential impact on meat quality. Keywords  Genic lncRNAs, lincRNA, Nellore cattle, RNA-Seq Consumers’ perception of meat quality products has evolved, becoming interested in the nutritional value of food, encompassing factors such as flavor, appearance, tenderness, food safety, health, social aspects, and sustainability1. Beef is a significant source of protein and is rich in essential polyunsaturated fatty acids (linoleic and linolenic acids), zinc, B vitamins, and iron, making it a unique and nutrient-rich food in the human diet to support healthy life, free from nutritional deficiencies2,3. Beef fatty acid profile plays a key role in beef ‘s quality traits, especially polyunsaturated fatty acids (PUFA), directly affecting sensory characteristics, such as taste, juiciness, and tenderness4. On the other hand, consumers are becoming more health-conscious and worried about the cholesterol and fats in food, mainly related to the 1Animal Science Department, School of Agricultural and Veterinary Sciences, São Paulo State University (Unesp), Via de Acesso Paulo Donato Castellane S/N, Departamento de Zootecnia, Jaboticabal 14884-900, SP, Brazil. 2Centre for Genetic Improvement of Livestock, Department of Animal Biosciences, University of Guelph, Guelph, ON, Canada. 3Nutrition and Animal Production Department, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, Avenida Duque de Caxias Norte, 225, Pirassununga 13635-900, SP, Brazil. 4National Council for Science and Technological Development, Brasilia 71605-001, DF, Brazil. email: bruna_salatta@hotmail.com; galvao.albuquerque@unesp.br OPEN Scientific Reports | (2025) 15:26109 1| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports http://www.nature.com/scientificreports http://crossmark.crossref.org/dialog/?doi=10.1038/s41598-025-11179-4&domain=pdf&date_stamp=2025-7-18 concentration of certain FAs, such as oleic acid, α-linolenic acid, conjugated linoleic acid (CLA), as well as omega-3 (ω3) and omega-6 (ω6), by its impact on human health status and diseases prevention5–8. In addition, meat fat has a high concentration of monounsaturated fatty acids (MUFA) with a low melting point, which can help reduce low-density lipoprotein (LDL) cholesterol concentration, refers as “bad” cholesterol, in blood circulation9. Although improving the beef FA profile can significantly enhance the quality of meat products, it is essential to ensure that the content of these FAs is adjusted to support long-term human health. Beef fatty acids are complex traits controlled by many genes and influenced by the environment10,11. The amount and type of fatty acids in beef meat depend on different factors, such as breed, nutrition, production system, sex, age, and carcass finishing level12. These factors influence the deposition and composition of FA, which limits the knowledge of the genetic mechanisms regulating these traits, hindering the genetic progress in producing healthier beef. Previous studies have reported that Nellore beef is nutritionally healthier than Angus beef due to higher amounts of ω3 and CLA13–17. In tropical production systems, Nellore cattle are widely used and exhibit considerable intra-breed variation in intramuscular fatty acid composition, particularly in the Longissimus thoracis (LT) muscle. This variability suggests the potential for genetic selection of animals with more favorable human health lipid profiles18–24. The deposition of FA in meat is a complex process involving the regulation of adipose metabolism (e.g., number and size of adipocytes) and the balance between lipogenesis and lipolysis25,26. This regulation involves multiple gene expressions, signal transduction, and network regulation, and new regulatory factors are constantly being identified, such as long non-coding RNAs (lncRNAs)26–28. Different authors have shown that lncRNA plays a crucial role in regulating protein-coding genes at different levels, such as epigenetic, transcriptional, and post-transcriptional regulation26,29–31. This regulation impacts the FA profile of meat by controlling the development of adipocytes, lipid metabolism, and fat-type conversion. However, there is a lack of information on lncRNA expression patterns, functions, complex gene networks, and molecular determinants related to different intramuscular FA profile deposition in the meat of cattle25,27,32–36. Hence, this study aims to identify differentially expressed lncRNAs from Nellore cattle muscle tissue with different FA profiles which may provide knowledge on the genetic and molecular mechanisms regulating FA profile to establish strategies for improving beef quality and directional selection. Results Phenotypic variation between groups Based on K-means analysis, three clusters with distinct FA profiles were identified (Fig. 1) and the descriptive FA content for each cluster are shown in Table 1. Cluster 1 (C1; n = 14 young bulls) exhibited the highest content for PUFA/SFA ratio, PUFA, ω6, ω3, linoleic acid and α-linolenic acid. The C3 was significantly higher in levels of saturated fatty acids (SFA), including stearic acid, myristic acid, and palmitic acid (p < 0.05; Table 10; Fig. 1). While Cluster 2 (C2; n = 24) presented an intermediate profile FA. Fig. 1.  The score plot showed individuals similarities for the fatty acid composition in Longissimus thoracis muscle of beef cattle. Scientific Reports | (2025) 15:26109 2| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports Differential expression analyses Differentially expressed long non-coding RNAs (lncRNA) annotated in the bovine genome reference (ARS.UCD 1.2) In the comparisons C1 vs. C2, C2 vs. C3, and C1 vs. C3, a total of 26 long non-coding RNAs were identified (Table  2). For the C1 vs. C2 comparison, one lncRNA, ENSBTAG00000050891, was upregulated, while five lncRNAs, ENSBTAG00000052746, ENSBTAG00000054148, ENSBTAG00000048982, ENSBTAG00000048541, ENSBTAG00000051111, ENSBTAG00000054291, and ENSBTAG00000052038, were downregulated in C1 relative to C2 (Table 2). Comparing C1 vs. C3, seven lncRNAs, ENSBTAG00000052922, ENSBTAG00000052100, ENSBTAG00000050348, ENSBTAG00000048918, ENSBTAG00000050739, ENSBTAG00000048899 and ENSBTAG00000052078, were upregulated, while three, ENSBTAG00000052746, ENSBTAG00000049466 and ENSBTAG00000048502, were downregulated in C1 relative to C3 (Table 2). In the C2 vs. C3 comparison, one lncRNA, ENSBTAG00000050891 was upregulated, whereas seven, ENSBTAG00000051111, ENSBTAG00000052922, ENSBTAG00000054148, ENSBTAG00000052100, ENSBTAG00000048544, ENSBTAG00000048918 and ENSBTAG00000052078 were downregulated in C2 relative to C3 (Table 2). All these lncRNAs were annotated as novel transcripts in the reference genome (ARS.UCD 1.2), however their functional roles are poorly characterized in the literature. Differentially expressed novel transcript length associated with long genic noncoding RNA annotated in the genome reference (ARS.UCD1.2). C1 vs. C2, 14 differentially expressed novel lncRNA were identified, whereby three were upregulated and eleven were downregulated for animals in C1 group compared with C2 group (Table 3). The DE lncRNAs were related to genes with functions on DNA repair, cellular response to lipid, RNA splicing, phosphorylation, regulation of MAPK cascade and regulation of B cell activation (Table 4). Comparing the group C1 vs. C3, 18 novels genic lncRNAs were found to be differentially expressed (Table 3), from those, eight were upregulated and ten were downregulated. The upregulated lncRNAs were associated with biological processes such as response to interleukin-1, central nervous system neuron differentiation, mitochondrion organization, and response to hypoxia (Table 5). Conversely, the downregulated lncRNAs were associated with genes involved in response to oxygen levels, mitochondrial transport, and transcription from RNA polymerase II promoters (Table 5). In comparing C2 vs. C3, 14 novel lncRNAs were pointed out as DE in the animals of the C2 group compared to the animals of the C3 group (Table 3). Among these 14 lncRNAs, five were upregulated lncRNA and were associated with activation of protein kinase activity, response to organic cyclic compound, lipid localization, lipid transport and regulation of cell proliferation (Table 6). While 9 lncRNAs were downregulated in C2 animals compared to C3 FA profile (Table 3). These were associated with genes with functions in transcription factor binding, actin cytoskeleton and immune response (Table 6). Differentially expressed novel long intergenic noncoding RNA (LincRNAs) In the C1 vs. C2 comparison, a total of 53 novel lincRNAs were identified, of which 17 were upregulated and 36 downregulated in C1 animals (Table 7; Supplementary Table S1;). The majority of these lincRNAs (56.60%) were in the antisense orientation relative to protein-coding transcripts. The upregulated lincRNAs were associated with genes involved in transcription factor binding, cellular response to lipids, positive regulation of cell proliferation, and mitochondrial transport (Table 4). Conversely, the downregulated novel lincRNAs were linked Category Fatty acids Formula SFA Myristic C14:0 SFA Palmitic C16:0 SFA Stearic C18:0 SFA Sum of SFA C4:0 + C6:0 + C8:0 + C10:0 + C11:0 + C12:0 + C13:0 + C14:0 + C15:0 + C16:0 + C17:0 + C18:0 + C21:0 + C24:0 MUFA Oleic C18:1 cis-9 MUFA Sum of MUFA C16:1 + C17:1 c10 + C18:1 t11 + C15:1 c10 + C20:1 c11 + C24:1 + C22:1 n9 + C18:1 c9 + C14:1 + 18:1n7 + C18:1 n9t PUFA Linoleic 18:2 cis-9,12 PUFA Conjugated Linoleic Acid (CLA) C18:2 cis9 trans11 PUFA Alpha-Linolenic C18:3 n3 PUFA Sum of ω3 C18:3 n3 + C20:3 n3 c11, c14, c17 + C22:6 n3 + C20:5 n3 PUFA Linoleic C18:2 cis9 cis12 n6 PUFA Sum of ω6 C18:3 n6 + C20:3 n6 c8, c11, c14 + C18:2 n6 + C20:4 n6 Ratio PUFA/SFA - Ratio ω6/ω3 - Table 1.  Classification and nomenclature of fatty acids analyzed in the study. Scientific Reports | (2025) 15:26109 3| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports to genes associated with the positive regulation of kinase activity, regulation of B cell activation, and DNA repair (Table 4). In the comparison between C1 and C3, a total of 85 DE lincRNAs were identified, of which 55 were upregulated and 30 downregulated in C1 (Table  8; Supplementary Table S2). Notably, the majority of these lincRNAs (65.88%) were located in the antisense orientation relative to their interacting genes. The upregulated lincRNAs were associated with genes involved in mRNA binding, lipid localization and transport, fatty acid metabolic processes, fat cell differentiation, transcription factor binding, and the negative regulation of cellular macromolecule biosynthetic processes (Table 5). Conversely, the downregulated lincRNAs were linked to genes associated with RNA biosynthetic processes, peptidyl-lysine modification, and ribonucleoside diphosphate metabolic processes (Table 5). Fifty-six DE lincRNA were identified in the C2 vs. C3 comparison, with 9 upregulated and 47 downregulated in C2 (Table 9; Supplementary Table S3). The majority of these lincRNAs (67.85%) were positioned in the antisense orientation relative to their interacting genes. The upregulated DE lincRNAs were linked to transcription factor binding, nuclear body organization, and the negative regulation of cell proliferation (Table 6). Conversely, the downregulated DE lincRNAs were associated with lipid localization, transport, positive regulation of cytoskeleton organization, actin cytoskeleton, and the regulation of cellular component size (Table 6). Functional analyses The genes associated with differentially expressed lncRNAs were functionally classified into molecular function, biological process, and cellular component categories. This classification was done for three cluster comparisons, namely C1 vs. C2, C1 vs. C3, and C2 vs. C3. For the C1 vs. C2 comparison, 41 significant GO terms (p-value < 0.05) were identified, which included 4 molecular functions (MF), 32 biological processes (BP), and 5 cellular components (CC) terms (Table 4). Similarly, for the C1 vs. C3 comparison, 145 significant GO terms were identified (p-value < 0.05), which included 27 molecular functions (MF), 98 biological processes (BP), and 20 cellular components (CC) terms (Table 5). For the C2 vs. C3 comparison, 61 significant GO terms Feature ID Position Lengtha p-Value FDR b FC (log2) c Comparison C1 vs. C2 ENSBTAG00000050891 16:55234389–55,238,149 341 1.41E-06 9.44E-05 11.17 ENSBTAG00000052746 21:32832866–32,857,979 647 6.44E-13 1.26E-10 -10.55 ENSBTAG00000054148 25:2533791–2,538,010 1840 3.25E-11 4.88E-09 -9.91 ENSBTAG00000048982 17:53500021–53,509,696 447 3.46E-07 2.66E-05 -8.40 ENSBTAG00000048541 10:2284385–2,291,785 564 6.59E-07 4.76E-05 -7.70 ENSBTAG00000051111 21:33076908–33,116,941 1347 1.31E-04 5.30E-03 -6.50 ENSBTAG00000054291 17:68750858–68,758,525 375 1.45E-04 5.78E-03 -11.63 ENSBTAG00000052038 23:50961431–50,974,711 2044 9.63E-04 2.68E-02 -4.47 Comparison C1 vs. C3 ENSBTAG00000052922 18:65702277–65,710,756 1048 1.27E-12 2.27E-10 10.60 ENSBTAG00000052100 18:65720235–65,729,032 4306 9.27E-10 9.68E-08 6.29 ENSBTAG00000050348 4:49799680–49,987,158 682 7.83E-07 4.48E-05 11.33 ENSBTAG00000048918 19:50079818–50,082,870 922 1.47E-04 4.98E-03 3.31 ENSBTAG00000050739 10:30457617–30,654,451 1039 3.71E-04 1.05E-02 11.41 ENSBTAG00000048899 7:21895844–22,018,311 1026 3.79E-04 1.07E-02 11.38 ENSBTAG00000052078 25:34334336–34,338,041 2032 8.26E-04 2.01E-02 4.39 ENSBTAG00000052746 21:32832866–32,857,979 647 3.14E-12 5.15E-10 -10.63 ENSBTAG00000049466 21:67740943–67,754,089 791 2.54E-05 1.02E-03 -9.30 ENSBTAG00000048502 9:99053414–99,081,437 923 8.51E-04 2.06E-02 -3.31 Comparison C2 vs. C3 ENSBTAG00000050891 16:55234389–55,238,149 341 1.52E-08 1.17E-06 14.82 ENSBTAG00000051111 21:33076908–33,116,941 1347 1.39E-14 3.19E-12 -8.79 ENSBTAG00000052922 18:65702277–65,710,756 1048 5.42E-11 6.97E-09 -8.92 ENSBTAG00000054148 25:2533791–2,538,010 1840 9.60E-09 7.61E-07 -9.75 ENSBTAG00000052100 18:65720235–65,729,032 4306 4.75E-08 3.35E-06 -4.92 ENSBTAG00000048544 23:36224038–36,465,322 1253 1.40E-06 7.11E-05 -10.90 ENSBTAG00000048918 19:50079818–50,082,870 922 4.39E-05 1.61E-03 -2.99 ENSBTAG00000052078 25:34334336–34,338,041 2032 3.23E-04 8.82E-03 -4.52 Table 2.  Differentially expressed long non-coding RNAs identified in Longissimus thoracis muscle of Nellore cattle with different fatty acid profiles. C - Cluster, a Transcript Length in bases pair; b False Discovery Rate; c FC (log2) = Fold change (log2). Scientific Reports | (2025) 15:26109 4| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports Feature ID Positon Length a p-Value FDR b FC (log2)c lncRNA annotated d Gene Interaction e Comparison (C1 vs. C2) lncRNA_18429.11 6:42982543–43,153,637 2562 5.44E-05 2.48E-03 2.48 ENSBTAT00000077875 ENSBTAG00000033327 lncRNA_5876.5 16:55234389–55,238,149 615 1.67E-04 6.49E-03 2.66 ENSBTAT00000081813 KLHL20, DARS2 ZBTB37, CENPL, RC3H1, SERPINC1 lncRNA_15786.3 3:58491541–58,579,144 480 2.14E-04 7.94E-03 13.91 ENSBTAT00000066768 CCN1, ZNHIT6 lncRNA_16505.6 4:41775570–41,794,763 1241 1.38E-12 2.55E-10 -7.55 ENSBTAT00000087015 - lncRNA_6086.4 16:79121509–79,142,756 3624 3.81E-12 6.57E-10 -6.54 ENSBTAT00000069212 ZNF281, KIF14 lncRNA_5039.5 8:97477285–97,504,848 5829 5.33E-12 9.08E-10 -4.94 ENSBTAT00000081266 - lncRNA_18428.3 6:42982543–43,153,637 692 3.00E-10 4.00E-08 -9.39 ENSBTAT00000077875 ENSBTAG00000033327 lncRNA_3567.2 13:37839904–37,856,732 8398 3.49E-10 4.59E-08 -4.66 ENSBTAT00000085977 ENSBTAG00000049520, ENSBTAG00000006043, BFSP1, PCSK2 DSTN lncRNA_12348.9 23:50961431–50,974,711 5505 7.12E-10 8.81E-08 -7.44 ENSBTAT00000076672 MYLK4, WRNIP1 lncRNA_7981.4 19:8790623–8,795,920 812 3.46E-07 2.66E-05 -8.40 ENSBTAT00000068745 VEZF1, SRSF1, DYNLL2, CUEDC1 lncRNA_19922.3 7:92888299–93,014,993 1228 1.98E-06 1.28E-04 -9.38 ENSBTAT00000067459 NR2F1, FAM172A lncRNA_10726.5 21:33076908–33,116,941 1161 1.73E-05 9.05E-04 -4.12 ENSBTAT00000082173 ENSBTAG00000024311, CSPG4, ODF3L1 lncRNA_17096.3 4:118297039–118,325,584 462 2.33E-05 1.17E-03 -9.11 ENSBTAT00000066814 LMBR1, MNX1, NOM1 lncRNA_16456.3 4:31551686–31,554,697 706 1.66E-04 6.47E-03 -11.45 ENSBTAT00000072688 ENSBTAG00000033806, IL6, TOMM7, FAM126A Comparison (C1 vs. C3) lncRNA_5876.4 16:55234389–55,238,149 615 8.24E-14 1.84E-11 8.03 ENSBTAT00000081813 KLHL20, DARS2, ZBTB37, CENPL, RC3H1, SERPINC1 lncRNA_6442.5 17:53500021–53,509,696 6591 3.05E-11 4.14E-09 5.71 ENSBTAT00000074572 SETD1B, CFAP251, PSMD9, ENSBTAG00000052582, TMEM120B lncRNA_6037.8 16:72109237–72,128,769 2734 6.70E-07 3.90E-05 10.85 ENSBTAT00000067279 RCOR3, TRAF5 lncRNA_5415.4 16:1072072–1,076,246 764 4.89E-06 2.28E-04 5.23 ENSBTAT00000086621 BTG2, CHI3L1 lncRNA_13894.1 26:51237158–51,241,924 3237 5.63E-06 2.59E-04 2.90 ENSBTAT00000073957 BNIP3 lncRNA_843.3 1:148950230–148,972,709 1213 4.96E-05 1.90E-03 3.05 ENSBTAT00000074316 SIM2 lncRNA_5415.2 16:1072072–1,076,246 985 9.50E-04 2.26E-02 3.27 ENSBTAT00000086621 BTG2, CHI3L1 lncRNA_12348.11 23:50961431–50,974,711 7845 1.34E-03 3.01E-02 10.18 ENSBTAT00000076672 MYLK4, WRNIP1 lncRNA_16505.6 4:41775570–41,794,763 1241 6.35E-13 1.21E-10 -8.52 ENSBTAT00000087015 - lncRNA_20382.10 8:70592566–70,718,220 1741 3.97E-12 6.38E-10 -10.54 ENSBTAT00000068948 SLC25A37, NKX3-1, LOXL2, ENTPD4 lncRNA_18428.3 6:42982543–43,153,637 692 5.38E-12 8.49E-10 -10.43 ENSBTAT00000077875 ENSBTAG00000033327 lncRNA_7981.4 19:8790623–8,795,920 812 1.49E-09 1.52E-07 -11.27 ENSBTAT00000068745 VEZF1, SRSF1, DYNLL2, CUEDC1 lncRNA_19922.3 7:92888299–93,014,993 1228 5.45E-06 2.51E-04 -10.36 ENSBTAT00000067459 NR2F1, FAM172A lncRNA_10726.5 21:33076908–33,116,941 1161 1.31E-05 5.59E-04 -4.63 ENSBTAT00000082173 ENSBTAG00000024311, ODF3L1, CSPG4 lncRNA_17096.3 4:118297039–118,325,584 462 2.10E-05 8.64E-04 -9.43 ENSBTAT00000066814 LMBR1, MNX1, NOM1 lncRNA_12504 23:36224038–36,465,322 604 2.41E-04 7.46E-03 -6.70 ENSBTAT00000083528 SOX4 lncRNA_20062.10 8:22841637–22,873,487 2286 3.72E-04 1.06E-02 -2.20 ENSBTAT00000070622 ENSBTAG00000048891, ENSBTAG00000054099, ENSBTAG00000055152, ENSBTAG00000050194, ENSBTAG00000052859, KLHL9, IFNAG, IFN-TAU lncRNA_15244.2 3:12245784–12,253,963 3500 1.76E-03 3.77E-02 -2.71 ENSBTAT00000072176 ENSBTAG00000024960 KIRREL1 Comparison (C2 vs. C3) lncRNA_20382.10 8:70592566–70,718,220 1741 2.96E-16 8.88E-14 10.38 ENSBTAT00000068948 SLC25A37, NKX3-1, LOXL2, ENTPD4 lncRNA_5415.4 16:1072072–1,076,246 764 1.93E-09 1.79E-07 8.12 ENSBTAT00000086621 BTG2, CHI3L1 lncRNA_9130.4 19:60774940–60,930,256 1149 3.50E-09 3.05E-07 3.36 ENSBTAT00000070427 - lncRNA_18429.11 6:42982543–43,153,637 2562 2.42E-06 1.17E-04 2.97 ENSBTAT00000077875 ENSBTAG00000033327 lncRNA_16618.6 4:55292087–55,307,803 5601 2.90E-04 8.09E-03 2.61 ENSBTAT00000070495 GPR85 lncRNA_6086.4 16:79121509–79,142,756 3624 2.06E-09 1.89E-07 -8.23 ENSBTAT00000069212 ZNF281, KIF14 lncRNA_6442.5 17:53500021–53,509,696 6591 2.95E-09 2.63E-07 -4.31 ENSBTAT00000074572 ENSBTAG00000052582, SETD1B, CFAP251, PSMD9, TMEM120B lncRNA_5876.4 16:55234389–55,238,149 615 4.57E-09 3.86E-07 -5.38 ENSBTAT00000081813 KLHL20, DARS2, ZBTB37, CENPL, RC3H1, SERPINC1 lncRNA_12348.9 23:50961431–50,974,711 5505 5.18E-09 4.33E-07 -7.90 ENSBTAT00000076672 MYLK4, WRNIP1 lncRNA_20062.7 8:22841637–22,873,487 2286 6.91E-07 3.79E-05 -11.23 ENSBTAT00000070622 ENSBTAG00000048891, ENSBTAG00000054099, ENSBTAG00000055152, ENSBTAG00000050194, ENSBTAG00000052859, IFN-TAU, KLHL9, IFNAG lncRNA_6037.8 16:72109237–72,128,769 2734 8.69E-07 4.65E-05 -9.89 ENSBTAT00000067279 RCOR3, TRAF5 Continued Scientific Reports | (2025) 15:26109 5| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports were identified (p-value < 0.05), which included 1 molecular function (MF), 47 biological processes (BP), and 13 cellular components (CC) terms (Table 6). Discussion The beef fatty acid profile contributes to its sensory properties and plays a significant role in determining the quality of the final product from a healthy perspective. Therefore, it is essential to select animals with genetic potential to produce healthier beef by increasing the levels of CLA and maintaining the optimal ratio of PUFA to SFA and ω6 to ω3. Aboujaoude et al.11 reported that it is feasible to select beef FA profile in Nellore cattle given there is additive genetic variation for most beef FA profiles (h2 varying from 0.01 to 0.35). This study revealed substantial variations (P < 0.05) across animals within the population, which was observed in the clustering analysis (Table  10). The C1 cluster exhibited higher levels of polyunsaturated fatty acids (PUFAs) (16.9 ± 2.03 g/100 g), including ω-6, ω-3, linoleic, and α-linolenic acids, compared with C2 (10.80 ± 1.74 g/100 g) and C3 (7.78 ± 1.68 g/100 g) (Table 10). Given the beneficial roles of PUFAs in human metabolism, C1 represents a promising group for selection in animal breeding programs. The C2 cluster exhibited an intermediate fatty acid profile, while C3 showed a lower PUFA/SFA ratio and higher concentrations of saturated fatty acids (SFA, 46.13 ± 2.00 g/100 g), monounsaturated fatty acids (MUFA, 39.19 ± 2.95 g/100 g), and conjugated linoleic acid (CLA, 0.34 ± 0.12 g/100 g) compared to the other groups - C1 (SFA: 42.91 ± 1.75 g/100 g; CLA: 0.17 ± 0.10 g/100 g) and C2 (SFA: 43.29 ± 1.46 g/100 g; CLA: 0.27 ± 0.09 g/100 g) (Table 10).The simultaneous presence of elevated SFA, MUFA and CLA content in cluster C3 may be associated with the activity of the Δ9 desaturase enzyme, which catalyzes the conversion of saturated fatty acids (SFA) into monounsaturated fatty acids (MUFA), and trans-vaccenic acid ( MUFA) into conjugated linoleic acid (CLA)37. This enzymatic activity suggests a metabolic interconnection and mutual dependence among these fatty acid classes. Furthermore, this cluster-based analysis reveals patterns of gene regulation across phenotypes, emphasizing coordinated lncRNA–gene networks rather than isolated gene effects. The genome produces numerous lncRNAs, which are transcripts with more than 200 nucleotides and do not encode protein-coding genes. Studies have indicated that the structure of lncRNAs is one of the most critical factors regulating various biological processes at the epigenetic, transcriptional, and translational levels38,39. When involved in transcriptional regulation, lncRNAs often function as ligands that interact with transcription factors, forming complexes that modulate gene transcription40. LncRNAs can be classified into several categories based on their genomic location, structure, and function. In this study, we categorized them according to their genomic location as genic lncRNAs (Table 2 and 3) and long intergenic non-coding RNAs (lincRNAs) (Tables 7, 8 and 9). Genic lncRNAs are those that overlap with protein-coding genes; they may intersect with exons, introns, or an entire gene, and can affect the functionality of their associated mRNA38. In contrast, lincRNAs are located in intergenic regions and do not overlap with protein-coding genes. These transcripts exhibit distinct features that differentiate them from mRNAs and are known to participate in chromatin remodeling, genome architecture modification, transcriptional regulation, and RNA stabilization41. In the present study, we identified 25 differentially expressed (DE) genic lncRNAs that are annotated in the bovine reference genome (ARS-UCD1.2), along with 46 DE novel transcripts associated with annotated lncRNAs, and 194 DE novel lincRNAs linked to fatty acid (FA) content in beef cattle. These lncRNAs are likely to exert regulatory influence over genes involved in fatty acid biosynthesis and lipid metabolism, thereby contributing to variations in fatty acid deposition in beef. Nonetheless, further functional analyses are needed to elucidate the specific biological roles and mechanisms of action of these lncRNAs. Differentially expressed genic long non-coding RNA Cluster 1 was characterized by a favorable lipid profile, enriched in polyunsaturated fatty acids (PUFA) - including ω6 to ω3 ratio and reduced levels of monounsaturated (MUFAs) and saturated fatty acids (SFAs), such as palmitic, stearic, and miristic acids. Several upregulated lncRNAs associated with this phenotype, including lncRNA_15786.3 (targeting CCN1), lncRNA_13894.1 (targeting BNIP3), and lncRNA_16456.3 (targeting IL6), may contribute to this metabolically advantageous fatty acid composition. In particular, the upregulation of lncRNA_15786.3 in Cluster 1 compared to Cluster 2, and its association with the CCN1 gene (Cellular Communication Network Factor 1) (Table  3), suggests a potential regulatory Feature ID Positon Length a p-Value FDR b FC (log2)c lncRNA annotated d Gene Interaction e lncRNA_3567.2 13:37839904–37,856,732 8398 1.01E-04 3.36E-03 -3.17 ENSBTAT00000085977 ENSBTAG00000049520, ENSBTAG00000006043, DSTN, BFSP, PCSK2 lncRNA_11324.5 22:36444667–36,922,968 1306 1.55E-04 4.84E-03 -11.06 ENSBTAT00000083930 ENSBTAG00000019048 lncRNA_843.3 1:148950230–148,972,709 1213 3.15E-04 8.64E-03 -2.59 ENSBTAT00000074316 SIM2 Table 3.  Novel transcript lengths associated with annotated long genic noncoding RNAs that are differentially expressed in the Longissimus thoracis muscle of Nellore cattle with distinct fatty acid profiles. a Transcript Length in basis pair, b False Discovery Rate, c FC (log2) = Fold change (log2), d transcript association = lncRNA annotated in the bovine reference annotation (ARS.UCD1.2), e Gene Interaction = Genes associated with annotated lncRNAs within the 200 kb window, with 100 kb upstream and 100 kb downstream, relative to the start and end positions of the gene, respectively. Scientific Reports | (2025) 15:26109 6| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports mechanism promoting PUFA deposition through modulation of lipogenic pathways. This regulation may involve downstream effects on key enzymes such as FASN (Fatty Acid Synthase), crucial for fatty acid synthesis, contributing to lipid formation and fat accumulation in cells42. Previous studies identified FASN gene variants associated with increased PUFA concentrations in milk43 and decreased SFA and MUFA levels in meat44aligning with the lipid profile observed in Cluster 1. GO: ID GOTerm p-value Nr. Genes * Genes lncRNA ID Biological process GO:0022411 cellular component disassembly 0.00 5 ARID2, DSTN, RUVBL1, SMN2, TOMM7 lincRNA_17358.2, lncRNA_3567.2, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lincRNA_10184.3, lincRNA_10184.1, lncRNA_16456.3 GO:0048545 response to steroid hormone 0.01 4 CNOT2, EIF4E, IL6, NR2F1 lincRNA_17393.3, lincRNA_18394.1, lncRNA_16456.3, lncRNA_19922.3 GO:0008284 positive regulation of cell proliferation 0.01 7 CD81, CCN1, HIPK1, IL6, KIF14, OSMR, SLC25A33 lincRNA_15009.1, lncRNA_15786.3, lincRNA_15543.1, lncRNA_16456.3, lncRNA_6086.4, lincRNA_10320.5, lincRNA_10320.8, lncRNA_10320.7, lincRNA_5714.2 GO:0014070 response to organic cyclic compound 0.01 6 CNOT2, DDX1, EIF4E, IL6, NR2F1, SMAD9 lincRNA_17393.3, lincRNA_2526.3, lincRNA_18394.1, lncRNA_16456.3, lncRNA_19922.3, lincRNA_3024.7, lincRNA_3024.5 GO:0050731 positive regulation of peptidyl-tyrosine phosphorylation 0.01 3 CD81, CSPG4, IL6 lincRNA_15009.1, lncRNA_10726.5, lncRNA_16456.3 GO:0006403 RNA localization 0.02 3 RUVBL1, SRSF1, ZNHIT6 lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lncRNA_7981.4, lncRNA_15786.3 GO:0071383 cellular response to steroid hormone stimulus 0.03 3 CNOT2, EIF4E, NR2F1 lincRNA_17393.3, lincRNA_18394.1, lncRNA_19922.3 GO:0009725 response to hormone 0.03 5 CNOT2, EIF4E, IL6, NR2F1, SLC25A33 lincRNA_17393.3, lincRNA_18394.1, lncRNA_16456.3, lncRNA_19922.3, lincRNA_5714.2 GO:0032147 activation of protein kinase activity 0.04 3 CD81, CSPG4, KIF14 lincRNA_15009.1, lncRNA_10726.5, lncRNA_6086.4 GO:0045860 positive regulation of protein kinase activity 0.04 4 CD81, CSPG4, CCN1, KIF14 lincRNA_15009.1, lncRNA_10726.5, lncRNA_15786.3, lncRNA_6086.4 GO:0043410 positive regulation of MAPK cascade 0.04 4 CD81, CSPG4, IL6, UNC5CL lincRNA_15009.1, lncRNA_10726.5, lncRNA_16456.3, lincRNA_11848.4 GO:0071396 cellular response to lipid 0.04 4 CNOT2, EIF4E, IL6, NR2F1 lincRNA_17393.3, lincRNA_18394.1, lncRNA_16456.3, lncRNA_19922.3 GO:0044255 cellular lipid metabolic process 0.04 4 AUH, CD81, ENSBTAT00000079956 lincRNA_20515.12, lincRNA_20515.13, lincRNA_20515.10, lincRNA_15009.1, lincRNA_20557.5 GO:0071407 cellular response to organic cyclic compound 0.05 4 CNOT2, EIF4E, NR2F1, SMAD9 lincRNA_17393.3, lincRNA_18394.1, lncRNA_19922.3, lincRNA_3024.7, lincRNA_3024.5 GO:0001558 regulation of cell growth 0.05 3 CCN1, KIF14, SLC25A33 lncRNA_15786.3, lncRNA_6086.4, lincRNA_5714.2 GO:0006281 DNA repair 0.05 4 DDX1, MMS22L, RUVBL1, WRNIP1 lincRNA_2526.3, lincRNA_20981.1, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lncRNA_12348.9, lncRNA_12348.11 GO:0006839 mitochondrial transport 0.05 3 RUVBL1, SLC25A33, TOMM7 lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lincRNA_5714.2, lncRNA_16456.3 GO:0050864 regulation of B cell activation 0.05 2 CD81, IL6 lincRNA_15009.1, lncRNA_16456.3 GO:0008380 RNA splicing 0.05 3 DDX1, SMN2, SRSF1 lincRNA_2526.3, lincRNA_10184.3, lincRNA_10184.1, lncRNA_7981.4 GO:0033674 positive regulation of kinase activity 0.05 4 CD81, CSPG4, CCN1, KIF14 lincRNA_15009.1, lncRNA_10726.5, lncRNA_15786.3, lncRNA_6086.4 Cellular Component GO:0036464 cytoplasmic ribonucleoprotein granule 0.00 4 CNOT2, DDX1, EIF4E, SMN2 lincRNA_17393.3, lincRNA_2526.3, lincRNA_18394.1, lincRNA_10184.3, lincRNA_10184.1 GO:0016604 nuclear body 0.02 6 DDX1, HIPK1, KLHL20, NOC3L, SMN2, SRSF1 lincRNA_2526.3, lincRNA_15543.1, lncRNA_5876.5, lncRNA_5876.4, lincRNA_13547.2, lincRNA_10184.3, lincRNA_10184.1, lncRNA_7981.4 Molecular function GO:0008134 transcription factor binding 0.01 6 ATOH8, CNOT2, EIF4E, RUVBL1, TLE4, ZNHIT6 lincRNA_2305.1, lincRNA_17393.3, lincRNA_18394.1, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lincRNA_20224.2, lncRNA_15786.3 GO:0004386 helicase activity 0.01 3 DDX1, RUVBL1, WRNIP1 lincRNA_2526.3, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lncRNA_12348.9, lncRNA_12348.11 GO:0016887 ATPase activity 0.01 5 DDX1, DYNLL2, KIF14, RUVBL1, WRNIP1 lincRNA_2526.3, lncRNA_7981.4, lncRNA_6086.4, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lncRNA_12348.9, lncRNA_12348.11 Table 4.  Gene ontology (GO) - Biological process, cellular component, and molecular Function - associated with genes interacting with differentially expressed LncRNAs and LincRNAs identified in the comparison between C1 and C2 fatty acid clusters. *Nr. Genes: Number of genes. Scientific Reports | (2025) 15:26109 7| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports GO: ID GOTerm p-value Nr. Genes* Genes lncRNAs Biological process GO:0006366 transcription from RNA polymerase II promoter 0.00 18 BRD3, CTCFL, E2F6, ELF2, GTF2A1, HOXC10, IER2, MNX1, NKX3-1, NR2F1, RCOR3, RUVBL1, SIM2, SMAD9, SOX4, TLE4, TRIM27, VEZF1 lincRNA_2832.1, lincRNA_3863.4, lincRNA_2540.2, lincRNA_2540.4, lincRNA_6258.4, lincRNA_6258.1, lincRNA_1840.2, lincRNA_17194.2, lincRNA_19092.2, lncRNA_17096.3, lncRNA_20382.10, lncRNA_19922.3, lncRNA_6037.8, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lncRNA_843.3, lincRNA_3024.7, lincRNA_3024.5, lncRNA_12504, lincRNA_20224.2, lincRNA_12128.6, lincRNA_12128.7, lncRNA_7981.4 GO:0021953 central nervous system neuron differentiation 0.00 5 ADARB1, BTG2, HOXC10, MNX1, SOX4 lincRNA_791.1, lncRNA_5415.4, lncRNA_5415.2, lincRNA_17194.2, lncRNA_17096.3, lncRNA_12504 GO:0032774 RNA biosynthetic process 0.00 27 BRD3, CTCFL, DDX1, E2F6, ELF2, GTF2A1, HOXC10, IER2, ENSBTAT00000053465, LOXL2, MLLT11, MNX1, MYT1L, NAB2, NKX3-1, NR2F1, RCOR3, RUVBL1, SIM2, SLC25A33, SMAD9, SMN2, SOX4, TLE4, TRAF5, TRIM27, VEZF1 lincRNA_2832.1, lincRNA_3863.4, lincRNA_2526.3, lincRNA_2540.2, lincRNA_2540.4, lincRNA_6258.4, lincRNA_6258.1, lincRNA_1840.2, lincRNA_17194.2, lincRNA_19092.2, lincRNA_7736.5, lincRNA_7736.1, lncRNA_20382.10, lincRNA_15403.1, lncRNA_17096.3, lincRNA_20692.2, lincRNA_17511.1, lincRNA_17511.6, lncRNA_19922.3, lncRNA_6037.8, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lncRNA_843.3, lincRNA_5714.2, lincRNA_3024.7, lincRNA_3024.5, lincRNA_10184.3, lincRNA_10184.1, lncRNA_12504, lincRNA_20224.2, lincRNA_12128.6, lincRNA_12128.7, lncRNA_7981.4 GO:0001501 skeletal system development 0.01 6 HOXC10, ITGB8, LOXL2, NAB2, SMAD9, SOX4 lincRNA_17194.2, lincRNA_16442.1, lncRNA_20382.10, lincRNA_17511.1, lincRNA_17511.6, lincRNA_3024.7, lincRNA_3024.5, lncRNA_12504 GO:0000959 mitochondrial RNA metabolic process 0.01 2 DARS2, SLC25A33 lncRNA_5876.4, lincRNA_5714.2 GO:0006839 mitochondrial transport 0.02 4 BNIP3, RUVBL1, SLC25A33, SLC25A37 lncRNA_13894.1, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lincRNA_5714.2, lncRNA_20382.10 GO:2,000,113 negative regulation of cellular macromolecule biosynthetic process 0.02 11 BTG2, E2F6, EIF4E, LOXL2, NAB2, NKX3-1, NR2F1, RCOR3, SIM2, TLE4, TRIM27 lncRNA_5415.4, lncRNA_5415.2, lincRNA_2540.2, lincRNA_2540.4, lincRNA_18394.1, lncRNA_20382.10, lincRNA_17511.1, lincRNA_17511.6, lncRNA_19922.3, lncRNA_6037.8, lncRNA_843.3, lincRNA_20224.2, lincRNA_12128.6, lincRNA_12128.7 GO:0010822 positive regulation of mitochondrion organization 0.02 3 BNIP3, MLLT11, RUVBL1 lncRNA_13894.1, lincRNA_15403.1, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3 GO:0001666 response to hypoxia 0.03 3 BNIP3, LOXL2, NKX3-1 lncRNA_13894.1, lncRNA_20382.10 GO:0010638 positive regulation of organelle organization 0.03 6 BNIP3, DCTN1, KIRREL1, MLLT11, RUVBL1, TRIM27 lncRNA_13894.1, lincRNA_2042.4, lncRNA_15244.2, lincRNA_15403.1, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lincRNA_12128.6, lincRNA_12128.7 GO:0021954 central nervous system neuron development 0.03 2 ADARB1, BTG2 lincRNA_791.1, lncRNA_5415.4, lncRNA_5415.2 GO:0006869 lipid transport 0.04 3 APOL3, NKX3-1, SPNS3 lincRNA_17681.1, lncRNA_20382.10, lincRNA_8294.10, lincRNA_8294.4 GO:0051495 positive regulation of cytoskeleton organization 0.04 3 DCTN1, KIRREL1, TRIM27 lincRNA_2042.4, lncRNA_15244.2, lincRNA_12128.6, lincRNA_12128.7 GO:0070482 response to oxygen levels 0.04 3 BNIP3, LOXL2, NKX3-1 lncRNA_13894.1, lncRNA_20382.10 GO:0010821 regulation of mitochondrion organization 0.04 3 BNIP3, MLLT11, RUVBL1 lncRNA_13894.1, lincRNA_15403.1, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3 GO:0032271 regulation of protein polymerization 0.04 3 DCTN1, KIRREL1, TRIM27 lincRNA_2042.4, lncRNA_15244.2, lincRNA_12128.6, lincRNA_12128.7 GO:0032535 regulation of cellular component size 0.04 4 KIRREL1, RAB22A, RAB5A, TRIM27 lncRNA_15244.2, lincRNA_3855.2, lincRNA_929.1, lincRNA_12128.6, lincRNA_12128.7 GO:0060070 canonical Wnt signaling pathway 0.05 2 RAB5A, SOX4 lincRNA_929.1, lncRNA_12504 GO:0010876 lipid localization 0.05 3 APOL3, NKX3-1, SPNS3 lincRNA_17681.1, lncRNA_20382.10, lincRNA_8294.10, lincRNA_8294.4 GO:0070555 response to interleukin-1 0.05 2 CHI3L1, NKX3-1 lncRNA_5415.4, lncRNA_5415.2, lncRNA_20382.10 GO:0008380 RNA splicing 0.05 4 DDX1, RBM38, SMN2, SRSF1 lincRNA_2526.3, lincRNA_3864.2, lincRNA_10184.3, lincRNA_10184.1, lncRNA_7981.4 GO:0018205 peptidyl-lysine modification 0.05 4 CTCFL, RUVBL1, SETD1B, SOX4 lincRNA_3863.4, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lncRNA_6442.5, lncRNA_12504 GO:0045444 fat cell differentiation 0.05 3 BNIP3, MEDAG, TMEM120B lncRNA_13894.1, lincRNA_3050.2, lncRNA_6442.5 GO:0071383 cellular response to steroid hormone stimulus 0.05 3 EIF4E, NKX3-1, NR2F1 lincRNA_18394.1, lncRNA_20382.10, lncRNA_19922.3 GO:0006631 fatty acid metabolic process 0.05 3 AUH lincRNA_20515.12, lincRNA_20515.13, lincRNA_20515.10 Cellular component GO:0030904 retromer complex 0.00 2 DCTN1, TRIM27 lincRNA_2042.4, lincRNA_12128.6, lincRNA_12128.7 Continued Scientific Reports | (2025) 15:26109 8| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports Cluster 2 exhibited an intermediate fatty acid profile, sharing characteristics of both C1 and C3. Although its molecular pattern was less pronounced, certain differentially expressed lncRNAs suggest a role in saturated fatty- acid (SFA) accumulation. For instance, lncRNA_11324.5, which is downregulated in Cluster 2 versus Cluster 3 (Table 3), targets the ENSBTAG00000019048 (Mediator Complex Subunit 28 - MED28) gene in humans. MED28 is ubiquitously expressed and has been shown to negatively regulate smooth muscle cell differentiation48,46, but its involvement in lipid metabolism remains unexplored47. Notably, genome‐wide association studies in cattle have linked MED28 to intramuscular fat (IMF) content and carcass traits48,49. Therefore, the reduced expression of lncRNA_11324.5 in C2 may reflect lower MED28 activity and consequently somewhat reduced SFA levels compared to Cluster 3 — while still exceeding those in Cluster 1. Similarly, lncRNA_19922.3, associated with NR2F1 (COUP transcription factor 1), was downregulated in both C1 vs. C2 and C1 vs. C3 comparisons (Table 3), suggesting a potential contribution to the lipid profile differences observed across clusters. The NR2F1 was enriched for GO biological function terms related to cellular lipid response (GO: 0071396) and to steroid hormone stimulus (GO:0071383) (Tables 4 and 5). NR2F1 is a sterol-binding transcription factor involved in the regulation of triglyceride synthesis and transport in enterocytes and has been implicated in lipid, cholesterol, and carbohydrate metabolism50–52. It has been validated as a candidate gene for intramuscular fat (IMF) deposition using multi-omics data from pig muscle tissues53,54. Moreover, a genomic association study in pigs conducted by Pena et al.55indicated NR2F1 within regions associated with palmitoleic acid, oleic acid, and MUFA. In the present study, C1 exhibited a lower total content of oleic acid and MUFA compared to C2 and C3 (Table 10), supporting the hypothesis that the reduced expression of lncRNA_19922.3 in C1 reflects lower NR2F1 activity and reduced MUFA levels. Additionally, lncRNA_20062.7 and lncRNA_20062.10, which interact with interferon-related genes such as IFN-TAU (Interferon Tau), IFNAG (Interferon-gamma), IFNA5 (Interferon Alpha 5), and IFNW1 (Interferon Omega 1) were downregulated in animals from C1 compared to C3, and in C2 compared to C3 (Table 3). These genes were enriched for GO biological processes related to immune activation, including cell activation involved in immune response (GO:0002263), leukocyte and lymphocyte activation (GO:0002366; GO:0002285) and adaptive immune response (GO:0002250) (Table 6). As members of the interferon family, these cytokines play central roles in immune regulation and inflammation56,57and they may also influence adipogenesis and fatty acid metabolism in muscle tissue58. In vitro studies have shown that oleic and palmitic acids can upregulate interferon-stimulated genes59while elevated levels of ω3 PUFA have been shown to suppress these pathways in mice60. The high concentrations of ω3 levels and reduced palmitic and oleic acid content observed in C1 and C2 may contribute to the reduced expression of interferon-associated lncRNAs in these groups. Cluster 1 exhibited upregulation of lncRNA_13894.1 relative to Cluster 2 and Cluster 3, which is associated with the BNIP3 gene. BNIP3 is implicated in mitochondrial β-oxidation and may influence the balance of ω6 and ω3 fatty acids61,62.Gene Ontology analysis revealed enrichment for fat-cell differentiation (GO:0045444) GO: ID GOTerm p-value Nr. Genes* Genes lncRNAs GO:0010494 cytoplasmic stress granule 0.01 2 DDX1, EIF4E lincRNA_2526.3, lincRNA_18394.1 GO:0034708 methyltransferase complex 0.01 3 E2F6, RUVBL1, SETD1B lincRNA_2540.2, lincRNA_2540.4, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lncRNA_6442.5 GO:0045335 phagocytic vesicle 0.02 2 RAB22A, RAB5A lincRNA_3855.2, lincRNA_929.1 GO:0015629 actin cytoskeleton 0.03 5 DCTN1, DYNLL2, IVNS1ABP, RAB22A, RAB5A lincRNA_2042.4, lncRNA_7981.4, lincRNA_5964.5, lincRNA_5964.1, lncRNA_5964.6, lincRNA_3855.2, lincRNA_929.1 GO:1,990,234 transferase complex 0.04 7 E2F6, GTF2A1, HOXC10, KLHL20, RUVBL1, SETD1B, VAC14 lincRNA_2540.2, lincRNA_2540.4, lincRNA_1840.2, lincRNA_17194.2, lncRNA_5876.5, lncRNA_5876.4, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lncRNA_6442.5, lincRNA_6802.5 GO:0035770 ribonucleoprotein granule 0.04 3 DDX1, EIF4E, SMN2 lincRNA_2526.3, lincRNA_18394.1, lincRNA_10184.3, lincRNA_10184.1 Molecular function GO:0003677 DNA binding 0.00 20 CTCFL, DDX1, E2F6, ELF2, GTF2A1, HOXC10, IER2, LBX2, LOC107132564, MNX1, NKX3-1, NR2F1, RCOR3, SIM2, SMAD9, SOX4, THAP9, TMPO, VEZF1, WRNIP1 lincRNA_3863.4, lincRNA_2526.3, lincRNA_2540.2, lincRNA_2540.4, lincRNA_6258.4, lincRNA_6258.1, lincRNA_1840.2, lincRNA_17194.2, lincRNA_19092.2, lincRNA_2036.1, lncRNA_17096.3, lncRNA_20382.10, lncRNA_19922.3, lncRNA_6037.8, lncRNA_843.3, lincRNA_3024.7, lincRNA_3024.5, lncRNA_12504, lincRNA_18674.6, lincRNA_18674.6, lincRNA_17577.2, lncRNA_7981.4, lncRNA_12348.9, lncRNA_12348.11 GO:1,990,837 sequence-specific double-stranded DNA binding 0.01 8 CTCFL, E2F6, HOXC10, IER2, NKX3-1, NR2F1, SOX4, VEZF1 lincRNA_3863.4, lincRNA_2540.2, lincRNA_2540.4, lincRNA_17194.2, lincRNA_19092.2, lncRNA_20382.10, lncRNA_19922.3, lncRNA_12504, lncRNA_7981.4 GO:0001067 regulatory region nucleic acid binding 0.01 9 CTCFL, E2F6, HOXC10, IER2, NKX3-1, NR2F1, RCOR3, SOX4, VEZF1 lincRNA_3863.4, lincRNA_2540.2, lincRNA_2540.4, lincRNA_17194.2, lincRNA_19092.2, lncRNA_20382.10, lncRNA_19922.3, lncRNA_6037.8, lncRNA_12504, lncRNA_7981.4 GO:0003729 mRNA binding 0.05 3 AUH, RBM38, SRSF1 lincRNA_20515.12, lincRNA_20515.13, lincRNA_20515.10, lincRNA_3864.2, lncRNA_7981.4 Table 5.  Gene ontology (GO) - Biological process, cellular component, and molecular Function - associated with genes interacting with differentially expressed LncRNAs and LincRNAs identified in the comparison between C1 and C3 fatty acid clusters. *Nr. Genes: Number of genes. Scientific Reports | (2025) 15:26109 9| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports GO: ID Term p-value Nr. Genes* Genes lncRNAs Biological process GO:0042100 B cell proliferation 0.00 4 CD180, CD81, IFN-TAU, IFNAG lincRNA_10210.6, lincRNA_15009.1, lncRNA_20062.10, lncRNA_20062.7 GO:0006898 receptor-mediated endocytosis 0.01 4 ATAD1, CD81, LOXL2, RAB5A lincRNA_13486.1, lincRNA_15009.1, lncRNA_20382.10, lincRNA_929.1 GO:0016197 endosomal transport 0.01 4 DCTN1, KLHL20, RAB5A, TRIM27 lincRNA_2042.4, lncRNA_5876.5, lncRNA_5876.4, lincRNA_929.1, lincRNA_12128.6, lincRNA_12128.7 GO:0032147 activation of protein kinase activity 0.01 4 CD81, CHI3L1, KIF14, NKX3-1 lincRNA_15009.1, lncRNA_5415.4, lncRNA_5415.2, lncRNA_6086.4, lncRNA_20382.10 GO:0042113 B cell activation 0.01 4 CD180, CD81, IFN-TAU, IFNAG lincRNA_10210.6, lincRNA_15009.1, lncRNA_20062.10, lncRNA_20062.7 GO:0046651 lymphocyte proliferation 0.01 4 CD180, CD81, IFN-TAU, IFNAG lincRNA_10210.6, lincRNA_15009.1, lncRNA_20062.10, lncRNA_20062.7 GO:0043549 regulation of kinase activity 0.01 7 ADARB1, CD81, CHI3L1, KIF14, NKX3-1, TRIM27, VAC14 lincRNA_791.1, lincRNA_15009.1, lncRNA_5415.4, lncRNA_5415.2, lncRNA_6086.4, lncRNA_20382.10, lincRNA_12128.6, lincRNA_12128.7, lincRNA_6802.5 GO:0043112 receptor metabolic process 0.01 3 ATAD1, CD81, RAB5A lincRNA_13486.1, lincRNA_15009.1, lincRNA_929.1 GO:0070661 leukocyte proliferation 0.02 4 CD180, CD81, IFN-TAU, IFNAG lincRNA_10210.6, lincRNA_15009.1, lncRNA_20062.10, lncRNA_20062.7 GO:0021953 central nervous system neuron differentiation 0.02 3 ADARB1, BTG2, HOXC10 lincRNA_791.1, lncRNA_5415.4, lncRNA_5415.2, lincRNA_17194.2 GO:0032535 regulation of cellular component size 0.02 4 DSTN, RAB22A, RAB5A, TRIM27 lncRNA_3567.2, lincRNA_3855.2, lincRNA_929.1, lincRNA_12128.6, lincRNA_12128.7 GO:0002285 lymphocyte activation involved in immune response 0.02 3 CD180, IFN-TAU, IFNAG lincRNA_10210.6, lncRNA_20062.10, lncRNA_20062.7 GO:0051495 positive regulation of cytoskeleton organization 0.03 3 DCTN1, DSTN, TRIM27 lincRNA_2042.4, lncRNA_3567.2, lincRNA_12128.6, lincRNA_12128.7 GO:0008285 negative regulation of cell proliferation 0.04 5 ADARB1, ARID2, ATOH8, BTG2, NKX3-1 lincRNA_791.1, lincRNA_17358.2, lincRNA_2305.1, lncRNA_5415.4, lncRNA_5415.2, lncRNA_20382.10 GO:0006869 lipid transport 0.04 3 APOL3, NKX3-1, SPNS3 lincRNA_17681.1, lncRNA_20382.10, lincRNA_8294.10, lincRNA_8294.4 GO:0002366 leukocyte activation involved in immune response 0.04 3 CD180, IFN-TAU, IFNAG lincRNA_10210.6, lncRNA_20062.10, lncRNA_20062.7 GO:0002263 cell activation involved in immune response 0.04 3 CD180, IFN-TAU, IFNAG lincRNA_10210.6, lncRNA_20062.10, lncRNA_20062.7 GO:0010638 positive regulation of organelle organization 0.05 5 CNOT2, DCTN1, DSTN, RUVBL1, TRIM27 lincRNA_17393.3, lincRNA_2042.4, lncRNA_3567.2, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lincRNA_12128.6, lincRNA_12128.7 GO:0033674 positive regulation of kinase activity 0.05 4 CD81, CHI3L1, KIF14, NKX3-1 lincRNA_15009.1, lncRNA_5415.4, lncRNA_5415.2, lncRNA_6086.4, lncRNA_20382.10 GO:0010876 lipid localization 0.05 3 APOL3, NKX3-1, SPNS3 lincRNA_17681.1, lncRNA_20382.10, lincRNA_8294.10, lincRNA_8294.4 GO:0014070 response to organic cyclic compound 0.05 5 CNOT2, IFN-TAU, IFNAG, NKX3-1, SMAD9 lincRNA_17393.3, lncRNA_20062.10, lncRNA_20062.7, lncRNA_20382.10, lincRNA_3024.7, lincRNA_3024.5 GO:0002250 adaptive immune response 0.05 3 IFN-TAU, IFNAG, TRIM27 lncRNA_20062.10, lncRNA_20062.7, lincRNA_12128.6, lincRNA_12128.7 GO:0034440 lipid oxidation 0.05 1 AUH lincRNA_20515.12, lincRNA_20515.13, lincRNA_20515.10 Cellular component GO:1,990,234 transferase complex 0.01 8 E2F6, GTF2A1, HOXC10, KLHL20, KLHL9, RUVBL1, SETD1B, VAC14 lincRNA_2540.2, lincRNA_2540.4, lincRNA_1840.2, lincRNA_17194.2, lncRNA_5876.5, lncRNA_5876.4, lncRNA_20062.10, lncRNA_20062.7, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3, lncRNA_6442.5, lincRNA_6802.5 GO:0015629 actin cytoskeleton 0.02 5 DCTN1, DSTN, IVNS1ABP, RAB22A, RAB5A lincRNA_2042.4, lncRNA_3567.2, lincRNA_5964.5, lincRNA_5964.1, lncRNA_5964.6, lincRNA_3855.2, lincRNA_929.1 GO:0035770 ribonucleoprotein granule 0.03 3 CNOT2, PCBP1, SMN2 lincRNA_17393.3, lincRNA_2417.4, lincRNA_10184.3, lincRNA_10184.1 GO:0005938 cell cortex 0.04 3 BFSP1, DCTN1, DSTN lncRNA_3567.2, lincRNA_2042.4 Molecular function GO:0008134 transcription factor binding 0.00 7 ATOH8, CNOT2, GTF2A1, NAB2, NKX3-1, RCOR3, RUVBL1 lincRNA_2305.1, lincRNA_17393.3, lincRNA_1840.2, lincRNA_17511.1, lincRNA_17511.6, lncRNA_20382.10, lncRNA_6037.8, lincRNA_11659.21, lincRNA_11659.1, lincRNA_11659.14, lincRNA_11659.17, lincRNA_11659.3 Table 6.  Gene ontology (GO) - Biological process, cellular component, and molecular Function - associated with genes interacting with differentially expressed LncRNAs and LincRNAs identified in the comparison between C2 and C3 fatty acid clusters. *Nr. Genes: Number of genes. Scientific Reports | (2025) 15:26109 10| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports lncRNAs located in sense direction a Feature ID Positon Lengthb p-Value FDR c FC(log2) d mRNA Interaction e Distance f Interaction location g lincRNA_20557.5 8:91054949–91,086,297 791 6.50E-13 1.26E-10 6.76 ENSBTAT00000079956 14,797 upstream lincRNA_7736.5 18:59685331–59,696,073 1112 2.60E-09 2.95E-07 6.23 ENSBTAT00000053465 78,735 upstream lincRNA_2305.1 11:49143024–49,147,255 855 4.03E-08 3.76E-06 3.13 ATOH8-201 16,527 upstream lincRNA_5255.1 15:65502793–65,506,317 2386 1.01E-04 4.25E-03 2.49 PDHX-201 170 downstream lincRNA_9933.3 2:126223556–126,230,654 1190 1.69E-04 6.54E-03 2.04 TRNP1-201 927 downstream lincRNA_10558.2 21:16690280–16,704,571 7132 3.04E-04 1.06E-02 3.26 ENSBTAT00000069947 17,948 downstream lincRNA_9517.5 2:52904578–52,913,931 3169 2.69E-21 1.44E-18 -9.83 ARHGAP15-201 51,209 downstream lincRNA_5265.1 15:65520195–65,531,625 1885 1.47E-19 6.62E-17 -5.83 PDHX-201 17,572 downstream lincRNA_11848.4 23:14861716–14,866,193 1681 1.02E-17 3.58E-15 -7.26 UNC5CL-201 85,367 downstream lincRNA_9517.4 2:52904578–52,913,931 3157 7.74E-17 2.50E-14 -5.91 ARHGAP15-201 51,221 downstream lincRNA_20515.12 8:86704251–86,724,738 3110 3.62E-10 4.74E-08 -6.88 AUH-203 18,194 downstream lincRNA_19092.2 7:12412390–12,426,648 2649 2.09E-09 2.40E-07 -3.82 IER2-201 7211 downstream lincRNA_2526.3 11:82614309–82,639,458 4315 2.48E-08 2.38E-06 -8.31 DDX1-201 38,762 downstream lincRNA_7736.1 18:59685331–59,696,073 1111 4.73E-06 2.79E-04 -2.86 ENSBTAT00000053465 78,735 upstream lincRNA_10320.5 20:35647442–35,665,372 2876 5.10E-05 2.35E-03 -4.52 OSMR-201 75,932 upstream lincRNA_4171.3 13:78409884–78,447,044 2212 1.67E-04 6.49E-03 -11.44 ENSBTAT00000079695 70,437 downstream lincRNA_13086.2 25:25769133–25,823,226 880 1.70E-04 6.58E-03 12.97 SBK1-201 27,121 upstream lincRNA_5964.5 16:66395474–66,405,633 1887 1.81E-04 6.90E-03 -4.48 IVNS1ABP-206 96,159 upstream lincRNA_2540.2 11:86418716–86,464,641 1033 4.45E-04 1.42E-02 -12.02 E2F6-201 16,736 downstream lincRNA_17103.9 5:5588820–5,627,943 3571 5.33E-04 1.65E-02 -11.12 PHLDA1-201 12,947 upstream lincRNA_10900.4 21:58589214–58,593,395 651 6.15E-04 1.86E-02 -2.42 CCDC197-201 10,826 upstream lincRNA_9841.2 2:120677247–120,682,616 3616 1.36E-03 3.55E-02 -2.02 AK2-201 55 downstream lincRNA_5964.1 16:66395474–66,405,633 1811 1.52E-03 3.87E-02 -4.90 IVNS1ABP-206 96,159 upstream lincRNAs located in antisense direction a Feature ID Positon Length b p-Value FDR c FC (log2) d mRNA Interaction e Distance f Interaction location g lincRNA_5578.3 16:29120343–29,123,464 2479 7.94E-21 4.11E-18 6.63 H3-3 A-201 32,484 downstream lincRNA_8796.3 19:44436479–44,443,655 1713 2.29E-08 2.21E-06 6.77 CCDC43-201 85 upstream lincRNA_17393.3 5:43266505–43,268,113 357 4.92E-08 4.51E-06 14.53 CNOT2-201 110 upstream lincRNA_5714.2 16:43978464–43,985,353 3333 6.05E-07 4.40E-05 2.89 SLC25A33-201 31,765 upstream lincRNA_8294.10 19:24908019–24,914,528 4663 1.52E-04 6.01E-03 2.23 SPNS3-201 22,427 upstream lincRNA_3119.2 12:34735808–34,750,812 3111 3.20E-04 1.11E-02 2.19 SGCG-201 29,925 upstream lincRNA_18383.11 6:24210504–24,526,275 3655 3.57E-04 1.20E-02 2.08 DDIT4L-201 360 upstream lincRNA_8294.4 19:24908019–24,914,528 4783 5.06E-04 1.58E-02 3.78 SPNS3-201 22,307 upstream lincRNA_15543.1 3:29450215–29,462,055 2414 1.20E-03 3.20E-02 3.32 HIPK1-201 269 upstream lincRNA_10184.3 20:10097610–10,105,934 1436 1.49E-03 3.82E-02 3.20 SMN2-201 44 upstream lincRNA_13547.2 26:15877179–15,880,904 1156 1.85E-03 4.55E-02 2.65 NOC3L-202 84 upstream lincRNA_15009.1 29:49217825–49,218,972 606 6.03E-19 2.58E-16 -5.78 CD81-201 1014 upstream lincRNA_20981.1 9:52492142–52,495,493 706 7.48E-19 3.15E-16 -9.70 MMS22L-202 1636 upstream lincRNA_17358.2 5:34560404–34,576,263 1840 2.78E-12 4.89E-10 -10.03 ARID2-202 41,974 upstream lincRNA_15447.1 3:20935933–20,944,419 2180 2.31E-09 2.64E-07 -10.74 MGC134040-201 6782 upstream lincRNA_12128.6 1:7104210–7,114,117 918 1.70E-07 1.41E-05 -6.87 TRIM27-201 88 upstream lincRNA_20224.2 8:55767546–55,825,069 1610 2.56E-07 2.05E-05 -10.80 TLE4-204 37,635 downstream lincRNA_280.3 1:69127181–69,127,765 425 2.91E-07 2.28E-05 -3.26 UMPS-201 102 upstream lincRNA_10919.2 21:60379945–60,383,584 1353 1.99E-06 1.29E-04 -3.19 SYNE3-201 92 upstream lincRNA_18394.1 6:25791826–25,795,075 1514 7.32E-05 3.21E-03 -4.56 EIF4E-202 86,079 downstream lincRNA_8476.5 19:33209590–33,211,733 801 1.74E-04 6.68E-03 -13.53 LRRC75A-202 367 downstream lincRNA_12128.6 23:30046878–30,066,974 1750 2.54E-04 9.14E-03 -12.48 TRIM27-201 1344 downstream lincRNA_14743.1 29:37152089–37,169,227 896 3.20E-04 1.11E-02 -11.32 CCDC86-201 515 upstream lincRNA_3024.7 12:24728114–24,731,701 742 3.72E-04 1.24E-02 -12.26 SMAD9-201 180 upstream lincRNA_6783.6 17:72362986–72,382,214 4197 7.54E-04 2.19E-02 -6.53 SLC7A4-201 605 upstream Continued Scientific Reports | (2025) 15:26109 11| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports (Table  5). The BNIP3 acts in different metabolic pathways, such as lipid metabolism61. Functionally, BNIP3 interacts with acetyl-CoA acyltransferase 2 (ACAA2), a mitochondrial enzyme catalyzing the terminal step of fatty acid β‑oxidation61,63. In sheep, single-nucleotide polymorphisms in ACAA2 have been linked to variations in the milk ω6/ω3 ratio64 and studies in fish have shown that dietary ω3 supplementation increases BNIP3 expression⁶⁶. Together, these data suggest that lncRNA_13894.1 may drive BNIP3 upregulation, supporting the ω6- and ω3-enriched lipid profile observed in Cluster 1 (Table 3). Conversely, lncRNA_16456.3, which targets the IL6 (Interleukin-6) gene, was downregulated in Cluster 1 versus Cluster 2 (Table 3). IL6 encodes a pro-inflammatory adipokine that modulates lipid homeostasis, fatty acid oxidation, and lipolysis in adipocytes and skeletal muscle65,66 and is enriched for cellular lipid response (GO:0071396) (Table 4). Interestingly, palmitic acid - a predominant saturated fatty acid - induces IL6 expression via inflammatory signaling pathways67–69. The reduced expression of lncRNA_16456.3 in C1 corresponds to diminished IL6 activity, consistent with the lower SFA levels in this group. However, IL6 has also been mapped near quantitative trait loci affecting PUFA, CLA, and SFA content in pigs, suggesting a pleiotropic role in fatty- acid composition70–74. Differentially expressed intergenic long non-coding RNA In Cluster 1, several lincRNAs were significantly downregulated in relation to C2 and C3, respectively, potentially reflecting attenuated stress and inflammatory signaling. Notably, lincRNA_18394.1 and lincRNA_2526.3 were both downregulated in C1 compared to C2 and C3 (Tables 7 and 8). These lincRNAs target the EIF4E (Eukaryotic Translation Initiation Factor 4E) and DDX1 (DEAD-Box Helicase 1) genes, respectively. The EIF4E was enriched to the “cellular response to lipid” (GO:0071396) GO term (Table 4). This gene is a key regulator of lipid homeostasis and energy metabolism in adipose tissue, coordinating fatty acid synthesis and oxidation75 and it is known to play a role in milk fatty acid synthesis in bovine mammary epithelial cells76. By contrast, DDX1 participates in DNA repair and mRNA transport77. Li et al.78 observed that the DDX1 protein binds to the untranslated region of insulin mRNA, inhibiting its translation, and that saturated fatty acids such as palmitic acid diminish insulin secretion in pancreatic β-cells, contributing to insulin resistance and diabetes79–83. Indeed, chronic exposure to SFAs is recognized as a driver of insulin resistance. Thus, the downregulation of lincRNA_18394.1 and lincRNA_2526.3 in Cluster 1 may indicate reduced activation of stress and insulin- resistance pathways, consistent with the lower total SFA levels observed in this group. In the comparison between Cluster 1 (C1) and Cluster 2 (C2), the differentially expressed lincRNA, lincRNA_17393.3 was significantly upregulated in C1 and is associated with the CNOT2 gene (Table 7). CNOT2 (CCR4-NOT Transcription Complex Subunit 2) is enriched for GO biological process terms related to hormonal response (GO:0009725) and cellular response to lipid (GO:0071396) (Table  4). This gene encodes a key component of the CCR4-NOT complex, which plays a pivotal role in post-transcriptional and transcriptional regulation, including mRNA degradation and translational control84,85. Functionally, CNOT2 has been implicated in adipocyte differentiation and fatty acids synthesis. Sohn et al.86 demonstrated that overexpression of CNOT2 in 3T3-L1 cells promotes adipogenesis and lipogenesis by upregulating transcription factors PPARγ and CEBPα, both of which are master regulators of lipid metabolism. Interestingly, several studies have reported that PPARγ and CEBPα expression is positively influenced by ω3 polyunsaturated fatty acids (PUFAs)87,88. Given the observed upregulation of lincRNA_17393.3 in C1 and the corresponding increase in total PUFA (including ω3) levels compared to C2, this lincRNA may contribute to the enhanced PUFA profile in C1 through regulation of CNOT2-mediated lipogenic pathways. In the C1 vs. C3 comparison, lincRNA_17194.2 was downregulated in C1 and was associated with the HOXC10 (Homeobox C10) gene (Table 8). HOXC10 has previously been implicated in key biological processes such as angiogenesis89fat metabolism90,91 and sexual regulation92. Kang et al.93 reported HOXC10 as a candidate gene lincRNAs located in antisense direction a Feature ID Positon Length b p-Value FDR c FC (log2) d mRNA Interaction e Distance f Interaction location g lincRNA_6371.2 17:51604344–51,756,900 1413 7.54E-04 2.19E-02 -2.42 CCDC92-201 756 upstream lincRNA_15447.4 3:20935933–20,944,419 1346 8.08E-04 2.31E-02 -2.26 MGC134040-201 6796 upstream lincRNA_18711.6 6:102631399–102,685,256 2552 1.00E-03 2.77E-02 -10.93 ENSBTAT00000008532 565 downstream lincRNA_11659.21 22:59504587–59,508,832 2979 1.78E-03 4.40E-02 -5.11 RUVBL1-201 221 upstream lincRNA_7507.1 18:52318710–52,407,580 2625 1.97E-03 4.78E-02 -2.97 ZNF180-203 3074 upstream Table 7.  Novel long intergenic noncoding RNAs differentially expressed in Longissimus thoracis muscle of Nellore cattle in the C1-fatty acid cluster compared to the C2-fatty acid cluster. a Direction of transcription of proximal RNA transcripts; b Transcript Length in basis pair; c FC (log2) = Fold change (log2); d False Discovery Rate; e transcript interaction = lincRNA gene overlaps with a transcript gene from the bovine reference annotation (ARS.UCD1.2). ; f Distance = distance between lincRNA and closest RNA transcript in the bovine reference annotation (ARS.UCD1.2); g Interaction location = are defined according to the type of interactions (genic or intergenic), assuming that intergenic lncRNA are not overlapping any RNA transcript (RNA/gene), then it can be further classified in: upstream (the lincRNA is upstream transcribed in head-to-head or tail-to- tail orientation with RNA partner or yet both same orientation) or downstream (the lincRNA is downstream transcribed in head-to-head or tail-to-tail orientation with RNA partner or yet both in same orientation). Scientific Reports | (2025) 15:26109 12| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports lncRNAs located in sense direction a Feature ID Positon Length (pb) b p-Value FDR c FC (log2) d mRNA Interaction e Distance f Interaction location g lincRNA_13486.1 26:9373528–9,377,974 2490 1.41E-20 1.01E-17 8.10 ATAD1-201 1300 downstream lincRNA_3864.2 13:58736919–58,750,531 1453 1.77E-14 4.32E-12 7.13 RBM38-201 7936 downstream lincRNA_20557.5 8:91054949–91,086,297 791 3.91E-13 7.73E-11 5.58 ENSBTAT00000079956 14,797 upstream lincRNA_10320.8 20:35647442–35,665,372 2557 4.37E-09 3.98E-07 9.41 OSMR-201 75,932 upstream lincRNA_5255.1 15:65502793–65,506,317 2386 3.61E-07 2.26E-05 3.91 PDHX-201 170 downstream lincRNA_10900.4 21:58589214–58,593,395 651 3.66E-07 2.28E-05 5.83 CCDC197-201 10,826 upstream lincRNA_10720.6 21:33142026–33,155,354 5446 3.95E-07 2.44E-05 8.44 ODF3L1-201 12,461 downstream lincRNA_2540.4 11:86418716–86,464,641 856 1.18E-06 6.39E-05 8.12 E2F6-201 16,909 downstream lincRNA_20515.13 8:86704251–86,724,738 2977 3.80E-06 1.83E-04 6.22 AUH-203 18,207 downstream lincRNA_3050.2 12:30041465–30,044,471 2760 1.04E-05 4.54E-04 3.70 MEDAG-201 26,845 upstream lincRNA_10558.1 21:16690280–16,704,571 7144 6.68E-05 2.49E-03 3.84 ENSBTAT00000069947 17,935 downstream lincRNA_10558.14 21:16690280–16,704,571 6381 3.34E-04 9.72E-03 12.04 ENSBTAT00000069947 18,323 downstream lincRNA_10558.9 21:16690280–16,704,571 6833 4.50E-04 1.23E-02 10.39 ENSBTAT00000069947 18,000 downstream lncRNA_10320.7 20:35647442–35,665,372 2557 6.73E-04 1.70E-02 3.85 OSMR-201 75,932 upstream lincRNA_16508.10 4:43223448–43,279,119 4572 1.14E-03 2.63E-02 11.50 PHTF2-201 41,821 downstream lincRNA_20515.10 8:86704251–86,724,738 3120 1.78E-03 3.81E-02 4.97 AUH-203 18,184 downstream lincRNA_9517.4 2:52904578–52,913,931 3157 1.66E-22 1.76E-19 -7.92 ARHGAP15-201 51,221 downstream lincRNA_5265.1 15:65520195–65,531,625 1885 1,55E-20 1,08E-17 -6,58 PDHX-201 17,572 downstream lincRNA_1878.2 10:102749910–102,772,451 4686 2.82E-17 1.09E-14 -6.47 U6-201 8839 downstream lincRNA_17681.1 5:74669803–74,711,373 1010 1.74E-11 2.49E-09 -5.55 APOL3-201 11,739 upstream lincRNA_20515.12 8:86704251–86,724,738 3110 3.79E-10 4.27E-08 -6.90 AUH-203 18,194 downstream lincRNA_19092.2 7:12412390–12,426,648 2649 1.69E-09 1.69E-07 -4.10 IER2-201 7211 downstream lincRNA_2526.3 11:82614309–82,639,458 4315 1.42E-06 7.54E-05 -8.61 DDX1-201 38,762 downstream lincRNA_5964.1 16:66395474–66,405,633 1811 7.94E-05 2.91E-03 -6.48 IVNS1ABP-206 96,159 upstream lincRNA_5964.6 16:66395474–66,405,633 1890 2.63E-04 8.01E-03 -4.09 IVNS1ABP-206 96,159 upstream lincRNA_16442.1 4:29193479–29,195,379 688 3.29E-04 9.59E-03 -2.59 ITGB8-201 77,720 downstream lincRNA_10720.7 21:33142026–33,155,354 2129 1.69E-03 3.63E-02 -3.79 ODF3L1-201 12,368 downstream lincRNA_7736.1 18:59685331–59,696,073 1111 1.82E-03 3.87E-02 -2.09 ENSBTAT00000053465 78,735 upstream lincRNA_9841.2 2:120677247–120,682,616 3616 2.36E-03 4.84E-02 -2.64 AK2-201 55 downstream lincRNAs located in antisense direction a Feature ID Positon Length (pb) b p-Value FDR c FC (log2) d mRNA Interaction e Distance f Interaction location g lincRNA_18738.3 6:108831172–108,833,800 2254 1.62E-21 1.43E-18 8.41 BOD1L1-201 165 upstream lincRNA_17511.1 5:56346812–56,351,225 553 8.71E-18 3.72E-15 9.25 NAB2-201 286 upstream lincRNA_16747.1 4:76603706–76,606,680 2756 2.08E-16 7.21E-14 7.95 CCM2-203 239 upstream lincRNA_18738.2 6:108831172–108,833,800 2257 6.65E-15 1.71E-12 6.13 BOD1L1-201 162 upstream lincRNA_18001.6 5:108915063–108,924,973 5266 2.19E-12 3.76E-10 8.62 CECR2-201 12,869 upstream lincRNA_10184.1 20:10097610–10,105,934 1477 2.29E-11 3.18E-09 7.74 SMN2-201 62 upstream lincRNA_10586.5 21:21519283–21,537,669 2678 8.21E-11 1.03E-08 11.66 ENSBTAT00000031184 2866 downstream lincRNA_6258.4 17:18453124–18,474,698 914 3.88E-10 4.36E-08 8.57 ELF2-201 147 upstream lincRNA_17577.2 5:62725526–62,731,661 1158 3.38E-09 3.16E-07 4.51 TMPO-201 12,982 downstream lincRNA_8796.1 19:44436479–44,443,655 1717 7.06E-09 6.25E-07 9.87 CCDC43-201 76 upstream lincRNA_15455.1 3:20557127–20,560,106 1103 7.88E-09 6.89E-07 10.62 VPS45-201 139 upstream lincRNA_2042.4 11:10233550–10,238,310 936 1.14E-08 9.67E-07 8.13 DCTN1-201 2229 upstream lincRNA_5578.3 16:29120343–29,123,464 2479 2.08E-07 1.37E-05 6.95 H3-3 A-201 32,484 downstream lincRNA_11659.1 22:59504587–59,508,832 3008 7.97E-07 4.54E-05 10.73 RUVBL1-201 189 upstream lincRNA_12128.7 23:30046878–30,066,974 1748 1.93E-06 9.93E-05 10.44 TRIM27-201 1344 downstream lincRNA_11659.14 22:59504587–59,508,832 3229 2.97E-06 1.47E-04 7.29 RUVBL1-201 187 upstream lincRNA_18674.6 6:97716507–97,750,095 1377 3.65E-06 1.77E-04 6.65 THAP9-201 2602 upstream lincRNA_3855.2 13:58045731–58,049,015 1933 7.19E-06 3.23E-04 3.17 RAB22A-201 101 upstream lincRNA_791.1 1:144934585–144,937,622 2739 7.97E-06 3.55E-04 4.48 ADARB1-201 204 upstream lincRNA_8476.6 19:33209590–33,211,733 1021 2.85E-05 1.14E-03 7.20 LRRC75A-202 367 downstream lincRNA_929.1 1:157196721–157,198,350 1337 5.73E-05 2.17E-03 4.94 RAB5A-201 151 upstream lincRNA_11659.17 22:59504587–59,508,832 3069 9.52E-05 3.42E-03 6.37 RUVBL1-201 188 upstream lincRNA_2036.1 11:10142822–10,146,171 2000 1.22E-04 4.23E-03 3.14 LBX2-201 2603 downstream lincRNA_8294.4 19:24908019–24,914,528 4783 1.70E-04 5.61E-03 12.95 SPNS3-201 22,307 upstream Continued Scientific Reports | (2025) 15:26109 13| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports influencing adipose deposition in fat-tailed sheep, while human study suggests its role in obesity and cholesterol regulation94,95. It is interesting to note that the presence of fatty acids can also modulate cellular cholesterol uptake and lipoprotein secretion. A study in humans showed that the presence of ω3 PUFAs reduced the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol synthesis96,97. Taken together, these data suggest that reduced expression of lincRNA_17194.2 in C1 may reflect lower HOXC10 activity in line with the high PUFA levels observed in C1 compared to Cluster 3. In both C1 vs. C3 and C2 vs. C3 comparison, the lincRNA, lincRNA_17681.1, was downregulated and was associated with the APOL3 (APOL-I to VI) gene (Tables 8 and 9). The APOL3 was enriched to GO-biological function terms related to lipid localization (GO: 0010876) and to lipid transport (GO:0006869) (Tables 5 and 6). APOL3 is known to be involved in cholesterol and sphingolipids transport and recycling in skeletal muscle in human98,99and in intramuscular fat (IMF) deposition in cattle100,101. The lower expression of lincRNA_17681.1 in C1 and C2 may reflect reduced activity of APOL3, consistent with the lower SFA levels (including myristic, palmitic, and stearic acids) observed in these clusters relative to C3. Together, these findings suggest that lincRNAs located in antisense direction a Feature ID Positon Length (pb) b p-Value FDR c FC (log2) d mRNA Interaction e Distance f Interaction location g lincRNA_3433.4 13:17510294–17,518,504 1169 1.72E-04 5.65E-03 12.94 IL15RA-201 121 upstream lincRNA_5714.2 16:43978464–43,985,353 3333 1.97E-04 6.32E-03 2.53 SLC25A33-201 31,765 upstream lincRNA_3024.5 12:24728114–24,731,701 896 2.16E-04 6.82E-03 12.14 SMAD9-201 165 upstream lincRNA_20692.2 8:111823798–111,834,775 3395 2.38E-04 7.38E-03 2.49 MYT1L-201 86,892 upstream lincRNA_8476.12 19:33209590–33,211,733 1027 2.57E-04 7.88E-03 12.76 LRRC75A-202 367 downstream lincRNA_17511.6 5:56346812–56,351,225 562 2.62E-04 7.99E-03 12.74 NAB2-201 1804 upstream lincRNA_18001.15 5:108915063–108,924,973 4906 2.85E-04 8.57E-03 12.62 CECR2-201 12,876 upstream lincRNA_6258.1 17:18453124–18,474,698 801 3.24E-04 9.48E-03 12.45 ELF2-201 1432 upstream lincRNA_18001.17 5:108915063–108,924,973 5263 4.86E-04 1.31E-02 3.22 CECR2-201 12,878 upstream lincRNA_1840.2 10:92511630–92,522,957 1195 7.11E-04 1.78E-02 3.83 GTF2A1-201 161 upstream lincRNA_11659.3 22:59504587–59,508,832 3272 8.38E-04 2.03E-02 11.46 RUVBL1-201 188 upstream lincRNA_15403.1 3:19689039–19,726,184 1166 1.28E-03 2.90E-02 11.14 MLLT11-201 410 downstream lincRNA_18674.5 6:97716507–97,750,095 1465 1.77E-03 3.79E-02 9.31 THAP9-201 1096 upstream lincRNA_17653.3 5:73720268–73,724,709 3062 2.17E-03 4.50E-02 2.30 RASD2-201 24,336 upstream lincRNA_18001.14 5:108915063–108,924,973 5266 2.24E-03 4.62E-02 3.19 CECR2-201 12,869 upstream lincRNA_20041.2 8:16267188–16,287,915 1056 1.77E-19 1.01E-16 -10.37 LINGO2-201 11,117 upstream lincRNA_20981.1 9:52492142–52,495,493 706 2.65E-19 1.45E-16 -10.80 MMS22L-202 1636 upstream lincRNA_17194.2 5:26047734–26,049,634 869 2.23E-12 3.81E-10 − 9.12 HOXC10-201 369 upstream lincRNA_20224.2 8:55767546–55,825,069 1610 3.43E-08 2.67E-06 -13.89 TLE4-204 37,635 downstream lincRNA_38.1 1:7104210–7,114,117 918 3.84E-07 2.38E-05 -8.67 CCT8-201 88 upstream lincRNA_15447.4 3:20935933–20,944,419 1346 1.87E-05 7.75E-04 -3.37 MGC134040-201 6796 upstream lincRNA_2832.1 11:104798248–104,801,001 2140 4.33E-05 1.68E-03 -2.47 BRD3-201 3413 downstream lincRNA_12128.6 23:30046878–30,066,974 1750 7.19E-05 2.66E-03 -14.61 TRIM27-201 1344 downstream lincRNA_14743.1 29:37152089–37,169,227 896 1.01E-04 3.58E-03 -13.40 CCDC86-201 515 upstream lincRNA_3024.7 12:24728114–24,731,701 742 1.53E-04 5.12E-03 -14.73 SMAD9-201 180 upstream lincRNA_6802.5 18:1343804–1,405,315 1285 1.41E-04 4.79E-03 12.72 VAC14-202 6224 upstream lincRNA_6783.6 17:72362986–72,382,214 4197 6.04E-04 1.56E-02 -7.45 SLC7A4-201 605 upstream lincRNA_18394.1 6:25791826–25,795,075 1514 8.06E-04 1.97E-02 -4.31 EIF4E-202 86,079 downstream lincRNA_3863.4 13:58694491–58,705,765 984 1.12E-03 2.60E-02 -2.57 CTCFL-201 11,892 downstream lincRNA_8796.4 19:44436479–44,443,655 1699 1.61E-03 3.50E-02 -2.61 CCDC43-201 98 upstream lincRNA_18001.20 5:108915063–108,924,973 5271 1.87E-03 3.95E-02 -4.23 CECR2-201 12,869 upstream lincRNA_10586.3 21:21519283–21,537,669 5164 2.28E-03 4.70E-02 -4.16 ENSBTAT00000031184 2866 downstream Table 8.  Novel long intergenic noncoding RNAs differentially expressed in Longissimus thoracis muscle of Nellore cattle in the C1-fatty acid cluster compared to C3-fatty acid cluster. a Direction of transcription of proximal RNA transcripts; b Transcript Length in basis pair; c FC (log2) = Fold change (log2); d False Discovery Rate; e transcript interaction = lincRNA gene overlaps with a transcript gene from the bovine reference annotation (ARS.UCD1.2). ; f Distance = distance between lincRNA and closest RNA transcript in the bovine reference annotation (ARS.UCD1.2); g Interaction location = are defined according to the type of interactions (genic or intergenic), assuming that intergenic lncRNA are not overlapping any RNA transcript (RNA/gene), then it can be further classified in: upstream (the lincRNA is upstream transcribed in head-to-head or tail-to- tail orientation with RNA partner or yet both same orientation) or downstream (the lincRNA is downstream transcribed in head-to-head or tail-to-tail orientation with RNA partner or yet both in same orientation). Scientific Reports | (2025) 15:26109 14| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports lincRNAs located in sense direction a Feature ID Positon Length b p-Value FDR c FC (log2) d mRNA Interaction e Distance f Interaction location g lincRNA_7736.5 18:59685331–59,696,073 1112 8.56E-11 1.05E-08 6.16 ENSBTAT00000053465 78,735 upstream lincRNA_9933.3 2:126223556–126,230,654 1190 5.22E-09 4.35E-07 3.61 TRNP1-201 927 downstream lincRNA_10558.2 21:16690280–16,704,571 7132 2.29E-07 1.41E-05 4.98 ENSBTAT00000069947 17,948 downstream lincRNA_2305.1 11:49143024–49,147,255 855 2.07E-06 1.01E-04 2.73 ATOH8-201 16,527 upstream lincRNA_13486.1 26:9373528–9,377,974 2490 5.24E-22 4.27E-19 -8.31 ATAD1-201 1300 downstream lincRNA_9517.5 2:52904578–52,913,931 3169 2.16E-19 1.17E-16 -9.76 ARHGAP15-201 51,209 downstream lincRNA_3864.2 13:58736919–58,750,531 1453 5.77E-16 1.65E-13 -7.39 RBM38-201 7936 downstream lincRNA_10900.4 21:58589214–58,593,395 651 7.42E-11 9.24E-09 -8.25 CCDC197-201 10,826 upstream lincRNA_17681.1 5:74669803–74,711,373 1010 6.50E-10 6.72E-08 -4.77 APOL3-201 11,739 upstream lincRNA_11848.4 23:14861716–14,866,193 1681 3.04E-09 2.70E-07 -6.31 UNC5CL-201 85,367 downstream lincRNA_10320.8 20:35647442–35,665,372 2557 5.36E-09 4.46E-07 -9.86 OSMR-201 75,932 upstream lincRNA_20515.13 8:86704251–86,724,738 2977 8.68E-08 5.83E-06 -7.23 AUH-203 18,207 downstream lincRNA_10720.6 21:33142026–33,155,354 5446 9.51E-08 6.34E-06 -8.51 ODF3L1-201 12,461 downstream lincRNA_5964.5 16:66395474–66,405,633 1887 4.68E-07 2.70E-05 -12.51 IVNS1ABP-206 96,159 upstream lincRNA_2540.4 11:86418716–86,464,641 856 4.46E-06 2.01E-04 -7.85 E2F6-201 16,909 downstream lincRNA_3470.1 13:23312186–23,369,059 4082 1.02E-04 3.38E-03 -2.32 ENSBTAT00000067334 45,318 downstream lincRNA_20515.10 8:86704251–86,724,738 3120 1.89E-04 5.67E-03 -5.74 AUH-203 18,184 downstream lincRNA_10558.14 21:16690280–16,704,571 6381 3.30E-04 8.99E-03 -11.93 ENSBTAT00000069947 18,323 downstream lincRNA located in antisense direction a Feature ID Positon Length b p-Value FDR c FC (log2) d mRNA Interaction e Distance f Interaction location g lincRNA_20041.2 8:16267188–16,287,915 1056 2.05E-09 1.89E-07 4.57 LINGO2-201 11,117 upstream lincRNA_17393.3 5:43266505–43,268,113 357 4.00E-08 2.85E-06 14.89 CNOT2-201 110 upstream lincRNA_5578.2 16:29120343–29,123,464 2479 3.23E-06 1.51E-04 2.96 H3-3 A-201 32,484 downstream lincRNA_2417.4 11:68352230–68,367,299 1440 8.63E-06 3.68E-04 4.14 PCBP1-201 44,166 upstream lincRNA_8294.10 19:24908019–24,914,528 4663 3.12E-04 8.59E-03 2.87 SPNS3-201 22,427 upstream lincRNA_7978.2 19:8080110–8,091,884 3121 3.53E-04 9.51E-03 13.23 MSI2-205 451 upstream lincRNA_18738.2 6:108831172–108,833,800 2257 1.33E-20 8.89E-18 -7.61 BOD1L1-201 162 upstream lincRNA_17511.1 5:56346812–56,351,225 553 1.86E-19 1.03E-16 -9.49 NAB2-201 286 upstream lincRNA_16747.1 4:76603706–76,606,680 2756 1.13E-17 4.34E-15 -8.66 CCM2-203 239 upstream lincRNA_15009.1 29:49217825–49,218,972 606 2.22E-13 4.30E-11 -6.76 CD81-201 1014 upstream lincRNA_17194.2 5:26047734–26,049,634 869 3.11E-13 5.92E-11 -9.73 HOXC10-201 369 upstream lincRNA_10184.1 20:10097610–10,105,934 1477 1.88E-11 2.67E-09 -7.02 SMN2-201 62 upstream lincRNA_2042.4 11:10233550–10,238,310 936 6.55E-11 8.23E-09 -8.15 DCTN1-201 2229 upstream lincRNA_10586.5 21:21519283–21,537,669 2678 7.73E-10 7.86E-08 -9.83 ENSBTAT00000031184 2866 downstream lincRNA_15455.1 3:20557127–20,560,106 1103 4.17E-09 3.56E-07 -9.59 VPS45-201 139 upstream lincRNA_3433.2 13:17510294–17,518,504 1039 1.80E-08 1.36E-06 -3.38 IL15RA-201 109 upstream lincRNA_8796.1 19:44436479–44,443,655 1717 1.16E-07 7.59E-06 -8.42 CCDC43-201 76 upstream lincRNA_17577.2 5:62725526–62,731,661 1158 3.57E-07 2.12E-05 -3.59 TMPO-201 12,982 downstream lincRNA_3855.2 13:58045731–58,049,015 1933 9.01E-07 4.80E-05 -3.24 RAB22A-201 101 upstream lincRNA_17358.2 5:34560404–34,576,263 1840 1.26E-06 6.49E-05 -9.96 ARID2-202 41,974 upstream lincRNA_11659.14 22:59504587–59,508,832 3229 1.62E-06 8.15E-05 -7.35 RUVBL1-201 187 upstream lincRNA_11659.1 22:59504587–59,508,832 3008 2.17E-06 1.05E-04 -10.27 RUVBL1-201 189 upstream lincRNA_12128.7 23:30046878–30,066,974 1748 3.26E-06 1.53E-04 -9.98 TRIM27-201 1344 downstream lincRNA_18674.6 6:97716507–97,750,095 1377 2.35E-05 9.10E-04 -5.76 THAP9-201 2602 upstream lincRNA_10919.2 21:60379945–60,383,584 1353 3.69E-05 1.38E-03 -6.04 SYNE3-201 92 upstream lincRNA_791.1 1:144934585–144,937,622 2739 4.16E-05 1.53E-03 -3.66 ADARB1-201 204 upstream lincRNA_11659.17 22:59504587–59,508,832 3069 4.95E-05 1.79E-03 -8.28 RUVBL1-201 188 upstream lincRNA_2036.1 11:10142822–10,146,171 2000 7.48E-05 2.56E-03 -3.11 LBX2-201 2603 downstream lincRNA_3024.5 12:24728114–24,731,701 896 1.02E-04 3.37E-03 -11.63 SMAD9-201 165 upstream lincRNA_929.1 1:157196721–157,198,350 1337 1.03E-04 3.39E-03 -4.11 RAB5A-201 151 upstream lincRNA_3433.4 13:17510294–17,518,504 1169 1.13E-04 3.68E-03 -14.02 IL15RA-201 121 upstream lincRNA_8476.14 19:33209590–33,211,733 514 1.24E-04 4.00E-03 -4.73 LRRC75A-202 367 downstream lincRNA_6802.5 18:1343804–1,405,315 1285 1.70E-04 5.23E-03 -12.54 VAC14-202 6224 upstream lincRNA_1840.2 10:92511630–92,522,957 1195 1.85E-04 5.58E-03 -3.74 GTF2A1-201 161 upstream lincRNA_18001.17 5:108915063–108,924,973 5263 2.73E-04 7.70E-03 -2.95 CECR2-201 12,878 upstream Continued Scientific Reports | (2025) 15:26109 15| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports lincRNA_17681.1 may serve as regulatory marker linked to SFA-rich phenotypes, with decreased expression supporting the leaner lipid profiles in C1 and C2. Overall, the enrichment analysis for the C2 vs. C3 comparison revealed gene ontology (GO) terms related to immune response, including cell activation involved in immune response (GO:0002263), adaptive immune response (GO:0002250), leukocyte activation involved in immune response (GO:0002366), lymphocyte activation involved in immune response (GO:0002285), and B cell activation (GO:0042113)  (Table 6). These findings align with the higher saturated fatty acid (SFA) content observed in both C2 and C3 clusters, which promote inflammation in adipose tissue through various of these mechanisms. SFAs activate pattern recognition receptors (PRRs) and toll-like receptor 4 (TLR4), leading to the production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL6)102–104. Moreover, SFAs induce endoplasmic reticulum stress and activate inflammatory signaling pathways, such as nuclear factor-kappa B (NF-κB), which further amplifies adipose tissue inflammation105. These biological processes are consistent with the immune- related GO enrichments detected in C2 and C3, suggesting a link between SFA accumulation and inflammatory activation. In this study, we identified several differentially expressed genic and intergenic lncRNAs associated with fatty acid composition in beef. While our findings provide new insights into the potential regulatory roles of lncRNAs in lipid metabolism and inflammatory processes, further functional studies are required to elucidate the specific mechanisms of action by which these lncRNAs interact with coding genes and modulate fatty acid metabolism in beef cattle. Understanding these interactions will be crucial for advancing genetic selection strategies aimed at improving the nutritional quality and health value of Nellore beef. Fatty acids Cluster 1 (n = 14) Cluster 2 (n = 24) Cluster 3 (n = 10) p-value PUFA/SFA 0.37a ± 0.05 0.24b ± 0.04 0.16c ± 0.03 5.97E-15 PUFA 16.19a ± 2.03 10.80b ± 1.74 7.78c ± 1.68 6.97E-15 ω6 10.19a ± 1.40 6.88b ± 1.33 5.07c ± 1.18 2.09E-12 ω3 5.72a ± 0.87 3.58b ± 0.58 2.33c ± 0.57 6.22E-16 Linolenic 9.37a ± 1.25 6.34b ± 1.22 4.69c ± 1.12 1.98E-12 α-Linolenic 0.94a ± 0.13 0.62b ± 0.11 0.46c ± 0.12 1.42E-12 ω6/ω3 1.79b ± 0.19 1.92b ± 0.25 2.18a ± 0.18 1.93E-04 CLA 0.17b ± 0.10 0.27a ± 0.09 0.34a ± 0.12 5.70E-04 Oleic 28.71b ± 2.03 33.18a ± 2.26 33.18a ± 2.26 3.04E-07 MUFA 33.88b ± 2.30 39.06a ± 2.32 39.19a ± 2.95 1.27E-07 SFA 42.91b ± 1.75 43.29b ± 1.46 46.13a ± 2.00 2.48E-05 Stearic 13.34b ± 1.64 13.96b ± 1.43 15.84a ± 1.68 7.06E-04 Miristic 1.54c ± 0.30 2.19b ± 0.26 2.71a ± 0.48 1.23E-10 Palmitic 19.69c ± 1.56 21.98b ± 0.99 23.59a ± 0.97 1.36E-09 Table 10.  Least square means of fatty acids (g/100 g of total fatty acids) in the Longissimus thoracis muscle of three clusters of Nellore bulls, grouped based on similarities in their fatty acid profiles. Data presented as mean and standard deviation. The concentration of fatty acids was expressed as a percentage of total fatty acid methyl esters. Means sharing different letters between columns were significantly different (p < 0.05) from one another according to Tukey’s test. lincRNA located in antisense direction a Feature ID Positon Length b p-Value FDR c FC (log2) d mRNA Interaction e Distance f Interaction location g lincRNA_6258.1 17:18453124–18,474,698 801 2.95E-04 8.20E-03 -11.43 ELF2-201 1432 upstream lincRNA_10210.6 20:12278005–12,356,729 2124 3.10E-04 8.53E-03 -12.26 CD180-201 61,290 upstream lincRNA_280.3 1:69127181–69,127,765 425 3.50E-04 9.45E-03 -3.12 UMPS-201 102 upstream Table 9.  Novel long intergenic noncoding RNAs differentially expressed in Longissimus thoracis muscle of Nellore cattle in the C2-fatty acid cluster compared to C3-fatty acid cluster. a Direction of transcription of proximal RNA transcripts; b Transcript Length in basis pair; c FC (log2) = Fold change (log2); d False Discovery Rate; e transcript interaction = lincRNA gene overlaps with a transcript gene from the bovine reference annotation (ARS.UCD1.2). ; f Distance = distance between lincRNA and closest RNA transcript in the bovine reference annotation (ARS.UCD1.2); g Interaction location = are defined according to the type of interactions (genic or intergenic), assuming that intergenic lncRNA are not overlapping any RNA transcript (RNA/gene), then it can be further classified in: upstream (the lincRNA is upstream transcribed in head-to-head or tail-to- tail orientation with RNA partner or yet both same orientation) or downstream (the lincRNA is downstream transcribed in head-to-head or tail-to-tail orientation with RNA partner or yet both in same orientation). Scientific Reports | (2025) 15:26109 16| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports Conclusion This study identified 38 novels genic lncRNAs and a total of 194 intergenic lncRNAs, highlighted lncRNA_15786.3, lncRNA_11324.5, lncRNA_13894.1, lncRNA_19922.3, and lincRNA_17393.3 that were interacting with genes essential to fatty acid metabolism. These interactions likely contribute to enhanced polyunsaturated (PUFA), ω3, ω6 and monounsaturated (MUFA) fatty acid profiles, consequently a healthier meat fat content. Specifically, these lncRNAs and lincRNAs target genes involved in adipogenesis (CCN1), muscle cell differentiation (MED28), β-oxidation (BNIP3), lipogenesis (CNOT2), and transport of triglycerides (NR2F1). Together, these findings provide valuable information about the complex regulatory network of lncRNAs, lincRNAs, and their gene partners affecting fatty acid composition in beef cattle, expanding our knowledge of noncoding regulatory elements associated with meat quality traits. This study lays the groundwork for future in-deep functional investigations aimed at unraveling the precise molecular mechanisms controlling FA deposition in bovine muscle. Materials and methods Ethics approval and consent to participate This study was approved to carry out procedures involving animals by ethics committee of the Faculty of Agrarian Sciences and Veterinary, Sao Paulo State University (UNESP) (certificate number 18340/16). Furthermore, all the data sampling was performed following the CEUA/ FCAV-UNESP guidelines and regulations according to Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, Brazil). In addition, we confirmed the statement that the study was conducted following the ARRIVE guidelines. Animals, samples collection and fatty acids composition A total of forty-eight young Nellore bulls belonging to the Capivara farm in São Paulo State, Brazil, were used. These animals were included in the Nellore Qualitas (QLT) commercial breeding program. During the growth phase, animals were raised under a grazing production system (Brachiaria sp. and Panicum sp.) and received mineral supplementation. The animals were finished in feedlots for 90 days, receiving a mixed diet based on corn silage and supplemented with concentrates based on sorghum grain and soybean meal in the proportion of concentrate/silage ratio (50/50 to 70/30). All animals belonged to the same contemporary group and were finished with the same nutritional management. The animals were slaughtered with an average age of 24 months and body weight of 550 kg in commercial slaughterhouses, following the Brazilian Federal Inspection Service procedures. RNA was extracted from Longissimus thoracis (LT) muscle tissue collected immediately after slaughter from the left half-carcass of each animal, between the 12th and 13th ribs. The samples were frozen in liquid nitrogen and stored at − 80 °C until further RNA-Seq analysis. After 48 h postmortem at 0–2 °C, the samples were collected from the Longissimus thoracis muscle (12–13th ribs of left half carcass) from each animal and stored at − 80 °C for posterior fatty acid (FA) assessment analyses. Beef FAs were extracted from the LT muscle samples (~ 100 g) according to the method described by Folch et al.107. The muscle samples were ground, and the lipids were extracted by homogenizing the sample with a solution of chloroform and methanol in the ratio of 2:1. Then NaCl was added at a concentration of 1.5% to isolate the lipids. The isolated lipids were subjected to methylation, and the resulting methyl esters were formed according to Kramer et al.108. The fatty acid composition was quantified using gas chromatography (GC-2010 Plus - Shimadzu AOC 20i auto-injector) equipped with an SP-2560 capillary column (100 m x 0.25 mm diameter with 0.02 mm thickness, Supelco, Bellefonte, PA), as described in Berton et al.18. The FA profile was quantified by normalizing the area under the curve of methyl esters using the GS Solution 2.42 software and then expressed as g/100 g of total FA of meat. From the identified FA profile, fourteen FAs were selected based on their health importance (seven individuals and seven groups of FAs) as described in Table 1. Animals clustering by amount of fatty acids The K-means method was used to classify 48 animals into three groups by their similarities in beef FA profiles (myristic, palmitic, stearic, oleic, linoleic, conjugated linoleic (CLA), α-linolenic and the groups of saturated fatty acids (SFA), monounsaturated (MUFA), polyunsaturated (PUFA), ω3, ω6, PUFA/SFA ratio and ω6/ω3). These three groups were determined using the gap statistics to compare the total intracluster variation for different values of k clusters with their expected values under the null reference distribution of the data. Normality for the FA profile was tested using Shapiro–Wilk’s normality test109and followed a normal distribution. The differences between the three groups on the FA profile were compared using the Least-Squares Means package in R110 and Tukey’s test (P < 0.05). RNA-sequencing Total RNA was isolated from 48 muscle tissue samples (~ 50 mg) using the RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s instructions. The extracted RNA’s purity was assessed by measuring its absorbance in a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). The quality of the total RNA extraction was evaluated using an Agilent 2100 Bioanalyzer, where only samples with RIN > 8 were used. The concentration and the presence of genomic DNA contamination were quantified using Qubit® 2. The mRNA paired-end libraries were created from each RNA sample using the Illumina TruSeq mRNA library preparation kit. Sequencing was conducted on the Illumina HiSeq 2500 platform to generate paired-end reads with 2 × 100 bp. Scientific Reports | (2025) 15:26109 17| https://doi.org/10.1038/s41598-025-11179-4 www.nature.com/scientificreports/ http://www.nature.com/scientificreports Quality control and reads alignment The FastQC program (Babraham Bioinformatics; Andrews S 2010. FastQC High Throughput Sequence QC Report)111 was used for quality control of the reads considering the following parameters: (1) quality scores, (2) GC-content, (3) N-content, (4) length distributions, (5) duplication, (6) overrepresented sequences, and (7) K-mer content. The raw reads were processed in two steps using Atropos (v1.1.19)112starting with the insert match algorithm, followed by the adapter match algorithm for the unprocessed reads that passed by the first step. Thereafter, the low-quality regions were also trimmed using “PRINSEQ-lite” software (v.0.20.3.;113). After filtering, HISAT2 (v.2.0.5)114 was used to map trimmed paired-end reads to the bovine genome reference (ARS-UCD1.2 Bos Taurus; ​h​t​t​p​:​/​​/​f​t​p​.​e​​n​s​e​m​b​l​​.​o​r​g​/​p​​u​b​/​c​u​​r​r​e​n​t​_​​f​a​s​t​a​/​​b​o​s​_​t​a​​u​r​u​s​/). Mapped reads were assembled using Cufflinks program (v2.2.1). Then, a matrix with transformed expression values was calculated by logarithm transformation (log2) and normalized using the cuffilinks packages (Cuffmerge/Cuffquant/Cuffnorm pipeline; ​h​t​t​p​:​/​​/​c​o​l​e​-​​t​r​a​p​n​e​​l​l​-​l​a​b​​.​g​i​t​h​​u​b​.​i​o​/​​c​u​f​f​l​i​​n​k​s​/​m​a​​n​u​a​l​/) with default settings. To ensure validity and reliability of any downstream analysis like DE, isoforms with no detectable expression (FPKM value “0” in all samples), or low expression (mean FPKM less than 0.01 in all samples) and transcripts that were shorter than 200 bp were filtered out115. Differential expression LncRNA analyses The LncDIFF is a powerful differential analysis tool for low abundance non-coding RNA expression data and was used to perform differential expression analysis115. The package “lncDIFF” was used with its parameter settings: link.function = “log,” simulated.pvalue = FALSE, permutation = 300 ​(​​​h​t​t​p​s​:​/​/​C​R​A​N​.​R​-​p​r​o​j​e​c​t​.​o​r​g​/​p​a​c​k​ a​g​e​=​l​n​c​D​I​F​F​​​​​)​. This package adopts the generalized linear model with zero-inflated exponential quasi-likelihood to estimate group effect on normalized counts and employs the likelihood ratio test in the differentially expressed genes. A pairwise group comparison (C1 vs. C2, C1 vs. C3 and C2 vs. C3) was used. Additionally, a Fold Change (FC) > | 2 |, p-value < 0.01 and FDR < 0.05 were used to filter DE transcripts and then identify and classify the DE lncRNAs. Identification of LncRNA Feelnc filter pipeline116 was used to identify potential long non-coding RNAs (lncRNAs) from 1,206,35 transcript models. Transcripts shorter than 200 bp, biotype-coding protein, single-exon transcripts, and biexonic transcripts with one exon size shorter than 25 bp were discarded117,118. After filtering, the FEELnc coding potential module (FEELnc codpot) was used to separate putative long noncoding RNAs (lncRNAs) from protein-coding RNAs by first computing a coding potential core (CPS, ranging from 0 to 1) for each transcript and then computing a CPS cut-off that maximizes both the lncRNA sensitivity and specificity using a tenfold cross-validation according to the input training files. This process helped classify the transcripts as putative lncRNAs or protein-coding RNAs27. The FEELnc classifier pipeline was also leveraged to classify each lncRNA with respect to its location and orientation compared to its closest annotated protein-coding genes. The FEELnc classifier module was used for possible function prediction of differentially expressed lncRNAs based on their nearest-neighbor protein-coding genes. The transcripts in this module are categorized in relation to the nearest RNA: Genic or Intergenic (lincRNA), with three subtypes that are the divergent, convergent and same-strand sub-classes, as detailed on the FEELnc website (https://github.com/tderrien/FEELnc). Additional information can be found in the FEElnc GitHub database (https://github.com/tderrien/FEELnc). This classification was applied to independently analyze the differentially expressed (DE) lncRNAs within each category. For novel transcript lengths associated with annotated lncRNAs, nearby genes located within a 200 kb window — defined as 100 kb upstream of the lncRNA start site and 100 kb downstream of its stop site — were annotated using the Ensembl Genome Browser (ARS-UCD1.2 assembly; ​h​t​t​p​s​:​/​/​u​s​e​a​s​t​.​e​n​s​e​m​b​l​.​o​r​g​/​B​ o​s​_​t​a​u​r​u​s​/​L​o​c​a​t​i​o​n​/​​​​​)​.​​ The ClueGO plug-in of the Cytoscape software was used to perform functional annotations of genes associated with DE lncRNAs, applying a p-value threshold of smaller than 5% for each group contrast. The ClueGO network was constructed using a kappa score < 0.05, to determine the strength of the association between genes and biological pathways. Data availability 44 RNAseq data samples are available in the publicly accessible Sequence Read Archive (SRA) database under the corresponding accession number PRJNA780472. Received: 19 January 2024; Accepted: 8 July 2025 References 1. Tonsor, G. T., Schroeder, T. 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