Transcriptome Analysis Reveals the Profile of Long Non-Coding RNAs during Myogenic Differentiation in Goats

Abstract

The long non-coding RNAs (lncRNAs) are emerging as essential regulators of the growth and development of skeletal muscles. However, little is known about the expression profiles of lncRNAs during the proliferation and differentiation of skeletal muscle satellite cells (MuSCs) in goats. In this study, we investigate potential regulatory lncRNAs that govern muscle development by performing lncRNA expression profiling analysis during the proliferation (cultured in the growth medium, GM) and differentiation (cultured in the differentiation medium, DM1/DM5) of MuSCs. In total, 1001 lncRNAs were identified in MuSC samples, and 314 differentially expressed (DE) (FDR < 0.05, |log2FC| > 1) lncRNAs were screened by pairwise comparisons from three comparison groups (GM-vs-DM1, GM-vs-DM5, DM1-vs-DM5). Moreover, we identified the cis-, trans-, and antisense-regulatory target genes of DE lncRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that these target genes were significantly enriched in muscle development-related GO terms and KEGG pathways. In addition, the network of interactions between DE lncRNAs and their target genes was identified, which included well-known myogenesis regulators such as Myogenic differentiation 1 (MyoD), Myogenin (MyoG), and Myosin heavy chain (MyHC). Meanwhile, competing endogenous RNA (ceRNA) network analysis showed that 237 DE lncRNAs could bind to 329 microRNAs (miRNAs), while miRNAs could target 564 mRNAs. Together, our results provide a genome-wide resource of lncRNAs that may contribute to myogenic differentiation in goats and lay the groundwork for future investigation into their functions during skeletal muscle development.

Keywords: differential expression; goats; lncRNAs; myogenic differentiation; transcriptome.

Figure 1

Morphological and gene expression characteristics of proliferating and differentiating goat MuSCs in vitro. (A) MuSCs were cultured in the growth medium until they achieved 80% confluence (GM, myoblasts). (B) MuSCs were cultured in the differentiation medium for 1 day (DM1, myocytes). (C) MuSCs were cultured in the differentiation medium for 5 days (DM5, myotubes). Scale bars = 100 µm. (D–F) Expression profiles of MyoD, MyoG, and MyHC from proliferation (GM) to differentiation (DM1 and DM5) in MuSCs. Data are the means ± SEM. * p < 0.05, ** p < 0.01.

Figure 2

Identification and characterization of lncRNA in goat MuSCs. (A) Type statistics of identified lncRNAs. (B) Chromosome distribution of identified lncRNAs. (C) The density distribution of lncRNAs was according to log10(FPKM). (D) The nine sample expressions in a violin plot, which was replaced by log10(FPKM).

Figure 3

Analysis of DE lncRNAs. (A) Numbers of up-regulated and down-regulated lncRNAs. (B) Venn diagram showing the DE lncRNAs at the three comparisons. (C–E) Hierarchical clustering analysis of DE lncRNAs through pairwise comparisons. Red: relatively high expression; Green: relatively low expression.

Figure 4

Enrichment analysis of cis-target genes of DE lncRNAs. (A) The top 26 GO terms of cis-target genes. (B) The top 20 KEGG pathways of cis-target genes. There are four circles from outside to inside. The first circle is the classification of GO or KEGG enrichment terms. Different colors represent different categories. The second circle shows the total number of foreground genes. The third circle represents the Q value and the number of background genes in the category. The fourth circle is the Rich Factor value of each category (the number of foreground genes in the category divided by the number of background genes). Each grid of the background auxiliary line represents 0.1.

Figure 5

Enrichment analysis of trans-target genes of DE lncRNAs. (A) The top 20 significant GO terms. (B) The top 20 significant KEGG pathways. The color of the circle represents the Q value. The size of the circle indicates the number of target genes.

Figure 6

The lncRNA-mRNA interaction networks. (A–C) LncRNA-mRNA networks for MSTRG.2787.1, MSTRG.8836.2, and MSTRG.8870.1. (D–F) LncRNA-mRNA networks associated with MyoD, MyoG, and MyHC in GM-vs-DM1, GM-vs-DM5, and DM1-vs-DM5. DE lncRNAs and their corresponding target genes were used to establish the lncRNA-gene interaction networks. In these networks, genes are displayed in purple arrow, while lncRNAs are displayed in circle; up-regulated lncRNA is indicated with red, while down-regulated lncRNA is indicated with blue. The cis-acting interactions are represented as dashed lines, whereas the trans-acting interactions are represented as solid lines.

Figure 7

The ceRNA networks. (A) CeRNA networks associated with MyoD and MyoG. (B–D) CeRNA networks for MSTRG.8836.2, MSTRG.2946.1, and MSTRG.12657.1. DE lncRNAs and mRNAs, as well as their common target miRNAs were used to establish the ceRNA networks. In these networks, genes are displayed in purple arrow, lncRNAs are displayed in yellow circle, and miRNAs are indicated with green diamond patterns.

Figure 8

Validation of eight lncRNAs by qRT-PCR. Expression levels were measured by RT-qPCR, calculated by the 2-ΔΔCt, and normalized to GAPDH. Data are the mean ± SEM.