LncRNA GTL2 regulates myoblast proliferation and differentiation via the PKA-CREB pathway in Duolang sheep

Abstract

Long non-coding RNAs (lncRNAs), which are RNA molecules longer than 200 nucleotides that do not encode proteins, are implicated in a variety of biological processes, including growth and development. Despite research into the role of lncRNAs in skeletal muscle development, the regulatory mechanisms governing ovine skeletal muscle development remain unclear. In this study, we analyzed the expression profiles of lncRNAs in skeletal muscle from 90-day-old embryos (F90), 1-month-old lambs (L30), and 3-year-old adult sheep (A3Y) using RNA sequencing. In total, 4 738 lncRNAs were identified, including 997 that were differentially expressed. Short-time series expression miner analysis identified eight significant expression profiles and a subset of lncRNAs potentially involved in muscle development. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the predicted target genes of these lncRNAs were primarily enriched in pathways associated with muscle development, such as the cAMP and Wnt signaling pathways. Notably, the expression of lncRNA GTL2 was found to decrease during muscle development. Moreover, GTL2 was highly expressed during the differentiation of skeletal muscle satellite cells (SCs) and was shown to modulate ovine myogenesis by affecting the phosphorylation levels of PKA and CREB. Additionally, GTL2 was found to regulate both the proliferation and differentiation of SCs via the PKA-CREB signaling pathway. Overall, this study provides a valuable resource and offers novel insights into the functional roles and regulatory mechanisms of lncRNAs in ovine skeletal muscle growth and development.

Keywords: LncRNA; PKA-CREB pathway; Sheep; Skeletal muscle; Skeletal muscle satellite cells.

Figure 1

Characterization of lncRNAs during muscle development A: Principal component analysis (PCA) of lncRNAs in nine samples. B: Venn diagram of lncRNA transcripts identified by Pfam, CPC, and CNCI. C: Distribution of mRNA and lncRNA exon numbers. D: Distribution of mRNA and lncRNA lengths. E: Distribution of mRNA and lncRNA ORF lengths.

Figure 2

Differentially expressed (DE) lncRNAs during muscle development A: Venn diagram of number of DE lncRNAs. B: Stacked histogram of number of DE lncRNAs across three comparison groups. C: Heatmap of 76 overlapping DE lncRNAs across three groups. D: GO enrichment analysis of genes potentially regulated by 76 overlapping DE lncRNAs. E: KEGG enrichment analysis of genes potentially regulated by 76 overlapping DE lncRNAs.

Figure 3

Short time-series expression miner (STEM) clustering analysis of differentially expressed (DE) lncRNAs A: Continuously up-regulated profile 12 ( P<0.05), where green line represents overall trend, and each colored line represents individual DE lncRNAs. B: KEGG analysis of genes potentially regulated by DE lncRNAs in profile 12. C: GO analysis of genes potentially regulated by DE lncRNAs in profile 12. D: Continuously down-regulated profile 3 ( P<0.05), where green line represents overall trend, and each colored line represents individual DE lncRNAs. E: KEGG analysis of genes potentially regulated by DE lncRNAs in profile 3. F: GO analysis of genes potentially regulated by DE lncRNAs in profile 3. Enriched terms and pathways are shown according to P-values.

Figure 4

Co-expression network of LncRNAs and their potentially regulated genes Red nodes represent lncRNAs, blue nodes represent their potentially regulated genes.

Figure 5

Identification of lncRNA GTL2 as a non-coding RNA A: Chromosomal location and length of lncRNA GTL2. Blue represents exon of GTL2, and yellow represents lncRNA GTL2. nt: nucleotide. B: GFP fusion plasmid construction for transfection, showing wild-type GFP gene (GFPwt) with the start codon ATGGTG and mutant GFP gene (GFPmut) with the start codon ATTGTT; lncRNA GTL2 ORF start codon ATG is mutated to ATT. C: Western blot analysis of GFP protein expression levels in 293T cells. D: RNA-seq analysis of lncRNA GTL2 expression in longissimus dorsi muscle across F90, L30, and A3Y stages. E: LncRNA GTL2 expression in longissimus dorsi muscle across three development stages (F90, L30, and A3Y). F: qPCR detection of lncRNA GTL2 in SCs during proliferation (GM) and differentiation (D1, D3, D5, and D7). Results are presented as mean±SEM, ^**: P<0.01; ^***: P<0.001.

Figure 6

LncRNA GTL2 inhibits proliferation of ovine skeletal muscle satellite cells (SCs) A: Knockdown of lncRNA GTL2 utilizing RNA interference (RNAi). B: CCK-8 assay showing cell vitality of SCs transfected with negative control or si-lncRNA GTL2. C: EdU assay detecting proliferation of SCs after knockdown of lncRNA GTL2. D: Numbers of cells in Gap 1 phase (G1), synthesis phase (S), and Gap 2 phase (G2) were calculated by flow cytometry of si-lncRNA GTL2. E: Western blot assay of CDK1 and CDK2 in SCs transfected with si-lncRNA GTL2. F: Cell transfection efficiency of over-lncRNA GTL2. G: CCK-8 assay showing cell vitality of SCs transfected with empty-pcDNA3.1(+) or over-lncRNA GTL2. H: EdU assay detecting proliferation of SCs after overexpression of lncRNA GTL2. I: Numbers of cells in Gap 1 phase (G1), synthesis phase (S), and Gap 2 phase (G2) were calculated by flow cytometry of over-lncRNA GTL2. J: Western blot assay of CDK1 and CDK2 in SCs transfected with over-lncRNA GTL2. Results are presented as mean±SEM, ^*: P<0.05; ^**: P<0.01; ^***: P<0.001.

Figure 7

LncRNA GTL2 promotes differentiation of ovine skeletal muscle satellite cells (SCs) A: Western blot assay of MyoG, MyoD, and SRF in SCs transfected with negative control or lncRNA GTL2 siRNA. B: Western blot assay of MyoG, MyoD, and SRF in SCs transfected with empty-pcDNA3.1(+) or over-lncRNA GTL2. C: Representative myotube staining of differentiated SCs transfected with si-lncRNA GTL2. Cells were stained with DAPI (blue) and MyHC (green) to visualize nuclei and myotubes, respectively. D: Immunofluorescence after transfection with over-lncRNA GTL2. SCs were differentiated for 3 days in differentiation medium. Results are presented as mean±SEM, ^*: P<0.05; ^**: P<0.01.

Figure 8

LncRNA GTL2 regulates proliferation and differentiation of ovine SCs via the PKA-CREB signaling pathway A: Western blot assay of PKA, p-PKA, CREB, and p-CREB in proliferating SCs transfected with negative control or lncRNA GTL2 siRNA. B: Western blot assay of PKA, p-PKA, CREB, and p-CREB in proliferating SCs transfected with empty-pcDNA3.1(+) or over-lncRNA GTL2. C: Western blot assay of PKA, p-PKA, CREB, and p-CREB in differentiating SCs transfected with negative control or lncRNA GTL2 siRNA. D: Western blot assay of PKA, p-PKA, CREB, and p-CREB in differentiating SCs transfected with empty-pcDNA3.1(+) or over-lncRNA GTL2. SCs were differentiated for 3 days in differentiation medium. Results are presented as mean±SEM, ^*: P<0.05.