Determining the Potential Roles of Branched-Chain Amino Acids in the Regulation of Muscle Growth in Common Carp (Cyprinus carpio) Based on Transcriptome and MicroRNA Sequencing

Abstract

Branched-chain amino acids (BCAAs) can be critically involved in skeletal muscle growth and body energy homeostasis. Skeletal muscle growth is a complex process; some muscle-specific microRNAs (miRNAs) are involved in the regulation of muscle thickening and muscle mass. Additionally, the regulatory network between miRNA and messenger RNA (mRNA) in the modulation of the role of BCAAs on skeletal muscle growth in fish has not been studied. In this study, common carp was starved for 14 days, followed by a 14-day gavage therapy with BCAAs, to investigate some of the miRNAs and genes that contribute to the regulation of normal growth and maintenance of skeletal muscle in response to short-term BCAA starvation stress. Subsequently, the transcriptome and small RNAome sequencing of carp skeletal muscle were performed. A total of 43,414 known and 1,112 novel genes were identified, in addition to 142 known and 654 novel miRNAs targeting 22,008 and 33,824 targets, respectively. Based on their expression profiles, 2,146 differentially expressed genes (DEGs) and 84 differentially expressed miRNA (DEMs) were evaluated. Kyoto Encyclopedia of Genes and Genome pathways, including the proteasome, phagosome, autophagy in animals, proteasome activator complex, and ubiquitin-dependent protein catabolic process, were enriched for these DEGs and DEMs. Our findings revealed the role of atg5, map1lc3c, ctsl, cdc53, psma6, psme2, myl9, and mylk in skeletal muscle growth, protein synthesis, and catabolic metabolism. Furthermore, miR-135c, miR-192, miR-194, and miR-203a may play key roles in maintaining the normal activities of the organism by regulating genes related to muscle growth, protein synthesis, and catabolism. This study on transcriptome and miRNA reveals the potential molecular mechanisms underlying the regulation of muscle protein deposition and provides new insights into genetic engineering techniques to improve common carp muscle development.

Figure 1

(a) Statistical map of mRNA functional classification. The abscissa indicates the database name, and the ordinate indicates the number or percentage of sequences annotated with that database. (b) Statistical volcano plot showing mRNA expression difference. Red and blue dots in the volcano plot indicate genes with significantly up- or downregulated expression, respectively, and gray dots represent genes with insignificant differences. The x-axis shows the log₂FC, and the y-axis shows the log₁₀p value. (c) Heat map of differential clustering of mRNA expression quantities. Red and blue represent the higher and lower gene expression in this sample, respectively; the left is a dendrogram of gene clustering and a module diagram of subclustering, and the name of the gene is indicated on the right; on the above is a dendrogram with clustering of samples, with name of the sample shown below.

Figure 2

Enrichment and analysis of DEG pathways in GO and KEGG samples. (a) Statistics of the top 20 GO terms enriched for DEGs between two groups. The vertical axis represents GO terms, the horizontal axis represents the rich factors, and the size of the dot represents the number of genes correlated with the GO term. The color of the dot corresponds to different p-adjusted ranges, and the smaller the enrichment p-adjust, the greater the significance. (b) Top-20 KEGG enrichment results. The vertical axis represents the path name, and the horizontal axis represents the richly factor. The size of the dot indicates the number of genes in this pathway, and the color of the dot corresponds to different p-adjusted ranges. (c) KEGG pathway enrichment chord diagram. Red stands for proteasome; blue represents autophagy-animal; green represents valine, leucine, and isoleucine degradation; purple stands for ubiquitin-mediated proteolysis; and pink stands for regulation of actin cytoskeleton in the picture, which denotes a significantly enriched pathway corresponding to differentially expressed genes, with genes on the left. The smaller the log₂FC, the greater the differential expression of multiple downregulated genes.

Figure 3

Analysis of DEMs. (a) Statistical chart of a useful read length distribution for small RNA sequencing. The abscissa represents the sequence length, and the ordinate indicates the number. (b) Statistical map of miRNA chromosome distribution. The abscissa is the chromosome number, and the ordinate is the number of sequenced reads that are located on the chromosome. (c) Target gene functional annotation summary statistical chart. The abscissa is the individual database name, and the ordinate is the number of annotations for the target gene in each database. (d) miRNA expression difference volcano plot. The ordinate value is the amount of expression of that gene in the treated sample. The red dots represent significantly upregulated miRNAs, the blue dots represent significantly downregulated miRNAs, the gray dots represent significantly differential miRNAs, and the point with a greater degree of deviation from the diagonal indicates a greater expression difference between the two samples.

Figure 4

Enrichment analysis of GO and KEGG pathways on the basis of DEMs. (a) GO enrichment analysis for target mRNAs of downregulated DEMs. The vertical axis represents the GO term, and the horizontal axis represents the richly factor. The size of the dots represents the number of genes in this GO term, and the color of the dots corresponds to different p-adjusted ranges. (b) KEGG enrichment analysis for target mRNAs of downregulated DEMs. The vertical axis indicates the KEGG pathway name, the horizontal axis indicates the rich factors, the size of the dot indicates how many genes are in this KEGG pathway, and the color of the dot corresponds to different p-adjusted ranges. (c) GO enrichment analysis for target mRNAs of upregulated DEMs. The vertical axis represents the GO term, and the horizontal axis represents the richly factor. The size of the dots represents the number of genes in this GO term, and the color of the dots corresponds to different p-adjusted ranges. (d) KEGG enrichment analysis for target mRNAs of upregulated DEMs. The vertical axis indicates the KEGG pathway name, the horizontal axis indicates the rich factors, the size of the dot indicates how many genes are in this KEGG pathway, and the color of the dot corresponds to different p-adjusted ranges.

Figure 5

Coexpression network of DEMs and DEGs. (a) Statistical network diagram of miRNA-mRNA relationships. Through the miRNA and mRNA interaction relationship, miRNA and target gene action pairs (miRNA-gene pairs) were visualized in a network form. Circular nodes represent genes, and diamond nodes represent miRNAs in the figure; internode lines (directed) represent the presence of targeting regulation between the two, green arrows represent positive regulation, and red short erector bars represent negative regulation. (b) Association network diagram of skeletal muscle growth-related genes and miRNAs. The ellipses in the figure represent the DEMs, the rectangles represent the DEGs, orange and red represent their expression quantities upregulated, and the opposite is blue that represents the expression quantity downregulated. Blue lines indicate positive correlations, and orange lines indicate negative correlation.

Figure 6

Transcriptome and small RNA sequencing data and real-time quantitative PCR (qRT-PCR) correlation validation map. Validation of the expression of mRNAs (a) and miRNAs (b) through qRT-PCR. The y-axis shows the relative expression (log₂FC). Bars represent the standard deviation of the three biological replicates.