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. 2023 Jul 3:11:1185823.
doi: 10.3389/fcell.2023.1185823. eCollection 2023.

Integrated analysis of circRNA, lncRNA, miRNA and mRNA to reveal the ceRNA regulatory network of postnatal skeletal muscle development in Ningxiang pig

Zonggang Yu  1 , Xueli Xu  1 , Nini Ai  1 , Kaiming Wang  1 , Peiwen Zhang  1 , Xintong Li  1 , Sui LiuFu  1 , Xiaolin Liu  1 , Jun Jiang  1 , Jingjing Gu  1 , Ning Gao  1 , Haiming Ma  1   2
Affiliations

Integrated analysis of circRNA, lncRNA, miRNA and mRNA to reveal the ceRNA regulatory network of postnatal skeletal muscle development in Ningxiang pig

Zonggang Yu et al. Front Cell Dev Biol. .

Abstract

Introduction: The development of skeletal muscle is regulated by regulatory factors of genes and non-coding RNAs (ncRNAs). Methods: The objective of this study was to understand the transformation of muscle fiber type in the longissimus dorsi muscle of male Ningxiang pigs at four different growth stages (30, 90, 150, and 210 days after birth, n = 3) by histological analysis and whole transcriptome sequencing. Additionally, the study investigated the expression patterns of various RNAs involved in muscle fiber transformation and constructed a regulatory network for competing endogenous RNA (ceRNA) that includes circular RNA (circRNA)/long non-coding RNA (lncRNA)-microRNA (miRNA)-messenger RNA (mRNA). Results: Histomorphology analysis showed that the diameter of muscle fiber reached its maximum at 150 days after birth. The slow muscle fiber transformation showed a pattern of initial decrease followed by an increase. 29,963 circRNAs, 2,683 lncRNAs, 986 miRNAs and 22,411 mRNAs with expression level ≥0 were identified by whole transcriptome sequencing. Furthermore, 642 differentially expressed circRNAs (DEc), 505 differentially expressed lncRNAs (DEl), 316 differentially expressed miRNAs (DEmi) and 6,090 differentially expressed mRNAs (DEm) were identified by differential expression analysis. Functions of differentially expressed mRNA were identified by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). GO enrichment analysis indicates that 40 known genes and 6 new genes are associated with skeletal muscle development. Additionally, KEGG analysis shows that these genes regulate skeletal muscle development via MAPK, FoxO, Hedgehog, PI3K-Akt, Notch, VEGF and other signaling pathways. Through protein-protein interaction (PPI) and transcription factor prediction (TFP), the action mode of skeletal muscle-related genes was explored. PPI analysis showed that there were stable interactions among 19 proteins, meanwhile, TFP analysis predicted 22 transcription factors such as HMG20B, MYF6, MYOD1 and MYOG, and 12 of the 19 interacting proteins were transcription factors. The regulatory network of ceRNA related to skeletal muscle development was constructed based on the correlation of various RNA expression levels and the targeted binding characteristics with miRNA. The regulatory network included 31 DEms, 59 miRNAs, 667 circRNAs and 224 lncRNAs. conclusion: Overall, the study revealed the role of ceRNA regulatory network in the transformation of skeletal muscle fiber types in Ningxiang pigs, which contributes to the understanding of ceRNA regulatory network in Ningxiang pigs during the skeletal muscle development period.

Keywords: Ningxiang pig; ceRNA network; circRNA; lncRNA; skeletal muscle development; whole transcriptome.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Detection of muscle fiber types of longissimus dorsi in Ningxiang pigs of different ages. (A–D) Real-time quantitative PCR analyzed the mRNA levels of MYHC IIa, MYHC IIb, MYHC IIx and MYHC I (E) The immunofluorescence staining of Longissimus dorsi muscle. The white scale bars represent 50 μm. Red indicates fast-twitch fibers and green indicates slow-twitch fibers. Immunofluorescence analyzed the percentage of slow-twitch fiber (F), fast-twitch fiber (G) and muscle fiber diameter (H). Data are presented as the mean ± SEM. n = 3. a, b, c Bar with different letter indicates significant difference (p < 0.05).
FIGURE 2
FIGURE 2
Upset analysis of four types of RNA by expression level screening. (A–B) upset of lncRNAs and miRNAs with TPM>0.05. (C) upset of mRNAs screened with TMP>1. (D) upset of circRNAs screened with RPM>0.05. Solid colored dots in the figure indicate presence in a stage. The number at the top of the bar represents the number of transcripts.
FIGURE 3
FIGURE 3
Paired difference analysis of four types of transcripts of Longissimus dorsi muscle of Ningxiang pigs at four postnatal stages (30d, 90d, 150d and 210 d). (A–D) mRNA, lncRNA, miRNA and circRNA, respectively. The number at the top of the bar chart indicates the number of upregulated or downregulated transcripts. The difference groups are above the center of the bar chart.
FIGURE 4
FIGURE 4
Functional annotation and enrichment analysis of differentially expressed mRNAs. (A) GO annotation of mRNA. GO enrichment (B), transcripts (C) and KEGG (D) related to skeletal muscle development during development. (E) Signal pathways involved in key genes. The size and color of the bubble represent the number of genes enriched in the term or pathway and enrichment significance, respectively. The chord width indicates the number of mRNAs, and the chord arrows indicate the terms or pathways involved.
FIGURE 5
FIGURE 5
PPI analysis of 40 skeletal muscle related proteins. (A) PPI analysis of 39 proteins in human. (B) PPI analysis of 39 proteins in mouse. (C) PPI analysis of 39 proteins in pig. (D) Protein Venn analysis of human, mouse and pig. (E) Venn analysis of the top 10 core proteins in human, pig and mouse. (F) Upset analysis of genes, transcription factors and PPI.
FIGURE 6
FIGURE 6
CircRNA/lncRNA-miRNA-mRNA interaction network analysis of differentially expressed genes related to skeletal muscle of Ningxiang pigs. (A) Venn analysis results of 49 differentially expressed genes associated with skeletal muscle and mRNA in ceRNA network. (B, C) Venn analysis results of differentially expressed circRNAs and lncRNAs in ceRNA networks, (D–G) Interaction network of skeletal muscle development marker genes (HMG20B, MYOD1, MYOG, MYF6). The pink and blue dots represent circRNA and lncRNA, respectively. Yellow triangles represent miRNA and green diamonds represent mRNA.
FIGURE 7
FIGURE 7
CeRNA interaction network and circRNA cyclization validation. (A–D) Verification of ceRNA network of four skeletal muscle related genes (HMG20B, MYOG, MYOD1, MYF6) by RT-qPCR. (E–F) Cyclization validation of Chr13_1453412_1456629 and Chr09_90892672_9089937. “face to face” and “back to back” are convergent primer and divergent primer amplification respectively. The arrow in the sanger sequencing peak diagram is back-splicing junction site. Data are presented as the mean ± SEM. n = 3.
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