Exploration of non-coding RNAs related to intramuscular fat deposition Xinjiang Brown cattle and Angus × Wagyu cattle

Abstract

Non-coding RNAs (ncRNAs) serve as crucial regulatory elements in the process of adipogenesis in animals; however, the specific roles and interrelationships of ncRNAs in bovine fat deposition remain poorly understood. This study aims to investigate the differentially expressed ncRNAs in the longissimus dorsi muscle of Xinjiang Brown cattle (XB) and Angus $\times$ Wagyu cattle (AW), to elucidate the regulatory mechanisms underlying lipidogenesis that may involve ncRNAs. Four Xinjiang Brown cattle and four Angus $\times$ Wagyu cattle were selected, ensuring they are subjected to identical feeding conditions, in order to evaluate the intermuscular fat (IMF) of longissimus dorsi muscles. The fat content of muscle tissue was quantified using the Soxhlet extraction method, revealing that the fat levels in the AW group were significantly elevated compared to those in the XB group. Taking muscle samples for paraffin sectioning and observing their morphology, it was found that the fat richness of the AW group was significantly higher than that of the XB group. Utilizing high-throughput RNA sequencing technology, we conducted an extensive transcriptomic analysis of longissimus dorsi muscles of XB and AW to identify significant ncRNAs implicated in fat metabolism and adipogenesis. The miRNA analysis yielded between 109,343,831 117,258,570 clean reads, whereas the lncRNA and circRNA analyses produced between 81,607,756 102,917,174 clean reads. Subsequent analysis revealed the identification of 53 differentially expressed miRNAs, 176 differentially expressed lncRNAs, and 234 differentially expressed circRNAs. KEGG enrichment analysis revealed that the target genes of differentially expressed miRNAs, lncRNAs, and circRNAs are significantly enriched in 2, 17, and 22 distinct pathways, respectively. The pathways associated with the differential enrichment of miRNA target genes involve processes such as phosphorylation and protein modification. Concurrently, the pathways linked to the varying enrichment of lncRNA target genes encompass G protein-coupled receptor signaling, regulation of cell death and apoptosis, activities related to GTPase activation, and functions governing nucleotide triphosphatases, among others. The circRNA exhibiting differential expression are significantly enriched in a variety of biological processes, including signal transduction, nucleic acid synthesis, cellular architecture, GTPase activation, and phosphatase activities, among others. The analysis of the ncRNA interaction network suggests that AGBL1, THRB, and S100A13 may play pivotal roles in the formation and adipogenic differentiation of adipocytes. In conclusion, we conducted a comprehensive analysis and discussion of the complete transcriptome of intermuscular fat tissue from the longissimus dorsi muscles in Xinjiang Brown cattle and Angus $\times$ Wagyu cattle. This study provides a theoretical foundation for enhancing our understanding of the molecular mechanisms underlying fat metabolism and deposition in beef cattle.

Keywords: Adipogenesis; Intermuscular fat; Non-coding RNA; Wagyu; Xinjiang brown cattle.

Fig. 1

IMF content and morphological observation of the longissimus dorsi muscle in XB and AW. A IMF content in the longissimus dorsi muscles of XB and AW. Results were expressed as mean ± standard deviation, n = 4. ****$p<0.001$. B Histological Analysis of Longissimus Dorsi Muscle in XB and AW. Hematoxylin eosin staining was used, the nucleus was dyed to blue, muscle fibers were dyed to red, adipose tissue is white. Magnification: 100$\times$

Fig. 2

The distribution of sequence lengths observed in the XB and AW libraries derived from circRNA sequencing results. Different colors represent the length distribution of circRNA in the longissimus dorsi muscle of various individuals

Fig. 3

The expression levels of three ncRNAs in XB and AW were analyzed. A miRNAs that are co-expressed in both XB and AW at the transcript level. B LncRNAs that are co-expressed in both XB and AW at the transcript level. C circRNAs that are co-expressed in both XB and AW at the transcript level. D The expression level distribution of miRNAs in XB and AW. E The expression level distribution of lncRNAs in XB and AW. F The expression level distribution of circRNAs in XB and AW

Fig. 4

A correlation heatmap illustrating the relationships among various samples. A Heatmap depicting miRNA correlation across samples. B Heatmap depicting lncRNA correlation across samples. C Heatmap depicting circRNA correlation across samples

Fig. 5

Differential expression miRNAs in the longissimus dorsi muscles between XB and AW. A Volcano plot illustrating the differential expression of miRNAs in XB and AW. The red dots indicate miRNAs that are upregulated ($p<0.05$), while the green dots denote downregulated miRNAs ($p<0.05$). The blue dots represent miRNAs exhibiting no significant expression differences. The dashed line illustrates the threshold for fold change in miRNAs expression. B Differentially expressed miRNAs between XB and AW

Fig. 6

Differential expression lncRNAs in the longissimus dorsi between XB and AW. A Volcano plot illustrating the differential expression of lncRNAs in XB and AW. The red dots indicate lncRNAs that are upregulated ($p<0.05$), while the green dots denote downregulated lncRNAs ($p<0.05$). The blue dots represent lncRNAs exhibiting no significant expression differences. The dashed line illustrates the threshold for fold change in gene expression. B Differentially expressed lncRNAs between XB and AW

Fig. 7

Differential expression circRNAs in the longissimus dorsi between XB and AW. A Volcano plot illustrating the differential expression of miRNAs in XB and AW. The red dots indicate circRNAs that are upregulated ($p<0.05$), while the green dots denote downregulated circRNAs ($p<0.05$). The blue dots represent circRNAs exhibiting no significant expression differences. The dashed line illustrates the threshold for fold change in circRNAs expression. B Differentially expressed circRNAs between XB and AW

Fig. 8

GO enrichment analysis of ncRNAs target genes. A GO enrichment of miRNA target genes. B GO enrichment of lncRNA target genes. C GO enrichment of circRNA target genes

Fig. 9

KEGG enrichment analysis of ncRNAs target genes. A KEGG enrichment of miRNA target genes. B KEGG enrichment of lncRNA target genes C KEGG enrichment of circRNA target genes

Fig. 10

The establishment and visualization of the LncRNA-miRNA-mRNA interaction network. A A network diagram illustrating the interactions among the target lncRNAs, miRNAs, and differentially expressed target genes across various combinations. B Assess the expression levels of downregulated lncRNAs, upregulated miRNAs, and their corresponding target genes. C Assess the expression levels of upregulated lncRNAs, downregulated miRNAs, and their corresponding target genes

Fig. 11

RT-qPCR verification of RNA-Seq results. We employed “log2(fold change)” as the ordinate, which signifies the fold change of the experimental group (AW) compare to the control group (XB), transformed into a logarithmic value with a base of 2