Exploring the miRNAs Profile in Dark-Cutting Beef

Abstract

In an animal production system, different stressors may cause the depletion of muscle glycogen stores, resulting in an elevated pH at 24 h post mortem (pH₂₄), which leads to cell metabolism alterations that affect the conversion of muscle into meat, causing meat quality defects, such as dark-cutting beef, also known as dark, firm, and dry (DFD) beef. This process may involve the alteration of small non-coding RNAs (miRNAs), which play critical regulatory roles in cellular processes. Here, we determined whether differential miRNA expression in the Longissimus thoracis et lumborum muscle from the Asturiana de los Valles breed at 24 h post mortem could serve as an early indicator of beef quality defects. Following total RNA extraction, complete miRNAome sequencing revealed 12 miRNAs that were significantly upregulated (p < 0.001) in DFD beef compared to the levels in CONTROL beef. These miRNAs are mainly involved in the cellular responses to redox imbalances and apoptosis. Among these, four miRNAs known to be related to oxidative stress (bta-miR-1246, bta-miR-2332, bta-miR-23b-5p, and bta-miR-2411-3p) were validated via quantitative RT-PCR. Some of their target proteins were also analyzed using Western blotting. High 70 kDa heat shock protein and low Caspase-9 expressions (p < 0.01) were found in DFD beef, suggesting the downregulation of apoptosis. These results suggest the importance of miRNAs in regulating stress in muscle cells during early post mortem, as differences in the abundance of some of these miRNAs are still observed at 24 h post mortem. These changes lead to an inadequate conversion of muscle into meat, resulting in meats with quality defects.

Keywords: DFD beef; MiRNA; meat quality; oxidative stress; skeletal muscle.

Figure 1

Post mortem evolution of Warner-Braztler shear force (WBSF) showing the meat tenderization pattern of CONTROL (blue line) and DFD (red line) samples. Different blue lowercase letters indicate significant differences in WBSF along post mortem for control samples (p < 0.05). Asterisks indicate significant differences between CONTROL and DFD at the same storage time. *** p < 0.001; ** p < 0.01.

Figure 2

MA plot of miRNA differential expression, showing the Log₂ fold change (y-axis) and the average expression (x-axis) across all samples. Significant changes in genes (>1 or <−1 Log₂ fold change) from DESeq2 (p < 0.05) are highlighted in color. Upregulated miRNAs are represented in red dots, while miRNAs with no significant changes in expression are represented in grey dots.

Figure 3

GeNorm analysis of qPCR-based candidate reference genes. (A) Genes with lower geNorm M values were considered more stable and the gene with an M value <0.5 was accepted as appropriate reference genes. Thus, miR-let7d-5p, miR-125b, and miR-10b were acceptable reference genes in this study. (B) The optimal number of reference genes required for normalization was determined based on pairwise variation (geNorm V value of n/n + 1) and a value <0.15 indicates the minimum number (n) of genes. Red line indicates the V value threshold. In this study, a combination of three reference genes was sufficient for normalization, as V3/4 was 0.139.

Figure 4

Expression levels of (A) bta-miR-1246, (B) bta-miR-2332, (C) bta-miR-23b-5p, and (D) bta-miR-2411-3p in CONTROL (grey bar) and DFD (white bar) beef samples were measured using RT-qPCR. The quantitative results are presented as mean ± SEM. **, p < 0.01.

Figure 5

Western blot analysis of proteins related to stress response and apoptosis markers in CONTROL (grey bar) and DFD (white bar) beef samples. Bar chart depicting the semiquantitative optical density (O.D.) (arbitrary units) of the blot bands of (A) 70 kDa heat shock protein of (HSP70) and (B) Caspase-9. (C) Representative immunoblots. Data are presented as mean ± SEM. **, p < 0.01.

Figure 6

microRNA functions and target genes in the post mortem skeletal muscle.