CircRNA profiling of skeletal muscle satellite cells in goats reveals circTGFβ2 promotes myoblast differentiation

Abstract

Background: Circular RNAs (circRNAs) function as essential regulatory elements with pivotal roles in various biological processes. However, their expression profiles and functional regulation during the differentiation of goat myoblasts have not been thoroughly explored. This study conducts an analysis of circRNA expression profiles during the proliferation phase (cultured in growth medium, GM) and differentiation phase (cultured in differentiation medium, DM1/DM5) of skeletal muscle satellite cells (MuSCs) in goats.

Results: A total of 2,094 circRNAs were identified, among which 84 were differentially expressed as determined by pairwise comparisons across three distinct groups. Validation of the expression levels of six randomly selected circRNAs was performed using reverse transcription PCR (RT-PCR) and quantitative RT-PCR (qRT-PCR), with confirmation of their back-splicing junction sites. Enrichment analysis of the host genes associated with differentially expressed circRNAs (DEcircRNAs) indicated significant involvement in biological processes such as muscle contraction, muscle hypertrophy, and muscle tissue development. Additionally, these host genes were implicated in key signaling pathways, including Hippo, TGF-beta, and MAPK pathways. Subsequently, employing Cytoscape, we developed a circRNA-miRNA interaction network to elucidate the complex regulatory mechanisms underlying goat muscle development, encompassing 21 circRNAs and 47 miRNAs. Functional assays demonstrated that circTGFβ2 enhances myogenic differentiation in goats, potentially through a miRNA sponge mechanism.

Conclusion: In conclusion, we identified the genome-wide expression profiles of circRNAs in goat MuSCs during both proliferation and differentiation phases, and established that circTGFβ2 plays a role in the regulation of myogenesis. This study offers a significant resource for the advanced exploration of the biological functions and mechanisms of circRNAs in the myogenesis of goats.

Keywords: Differential expression; Differentiation; Goat; circRNA sequencing; circTGFβ2.

Fig. 1

Identification and characteristics of circRNAs in goat skeletal muscle satellite cells (MuSCs). (A) Schematic illustration of the experimental design. (B) Venn analysis of circRNAs detected at each time point. (C) Principal component analysis based on the expression of circRNAs. (D) Types of all circRNAs

Fig. 2

Analysis of DEcircRNAs in goat MuSCs across different stages. (A) Numbers of up-regulated and down-regulated circRNAs in goat MuSCs across three stages. (B) Venn diagram showing the DEcircRNAs at the three comparisons. (C) Hierarchical clustering heat map of all DEcircRNAs. Data are expressed as RPM. Red represents relatively high expression, blue represents relatively low expression

Fig. 3

Functional enrichment analysis for host genes of differentially expressed circRNAs. (A) The top 10 significance terms of biological process in DM1 vs. DM5. (B) The top 10 significance terms of biological process in GM vs. DM1. (C) The top 10 significance terms of biological process in GM vs. DM5. (D) The top 10 significance pathways in DM1 vs. DM5. (E) The top 10 significance pathways in GM vs. DM1. (F) The top 10 significance pathways in GM vs. DM5

Fig. 4

The circRNA-miRNA interaction networks, including 21 DEcircRNAs and 47 miRNAs. Blue represent DEcircRNAs and yellow represent miRNAs

Fig. 5

Experimental validation of circRNAs. (A) Sanger sequencing shows head-to-tail junctions. Blue arrow shows back spliced junctions. (B) Agarose gel electrophoresis test for PCR product of six circRNAs using different templates. gDNA represent genomic DNA; R + indicates the samples were treated with exonuclease. (C) Expression profiles of the six randomly selected circRNAs based on RNA-Seq and qRT-PCR

Fig. 6

The characterization and function of circTGFβ2 in MuSC differentiation. (A) The back-site junction of circTGFβ2 was identified using a divergent primer and sequenced by Sanger sequencing. (B) Agarose gel electrophoresis test for PCR product of circTGFβ2 using different templates. Random shows the cDNA template synthesized using random primers for RT-PCR, while Oligo(dT) denotes the cDNA synthesized using Oligo(dT) primers; gDNA represent genomic DNA; RNase R + indicates the samples were treated with exonuclease. (C) qRT-PCR analysis of the expression of circTGFβ2 and TGFβ2 in goat myoblasts treated with RNase R. (D) The abundance of circTGFβ2 in various tissues of goats on the third day after birth. (E) The expression level of circTGFβ2 during proliferation and differentiation of goat skeletal muscle satellite cells. (F) RNA FISH assay was performed to determine the subcellular localization of circTGFβ2 in goat skeletal muscle cells, blue indicates nuclei stained with DAPI; red indicates RNA probes recognizing circTGFβ2. Scale bar, 10 μm. (G) Luciferase reporter activity of circTGFβ2 in MuSCs co-transfected with chi-miR-206 mimics or mimics NC. (H) Luciferase reporter activity of circTGFβ2 in MuSCs co-transfected with chi-miR-211 mimics or mimics NC. (I) CircTGFβ2 was successfully overexpressed in MuSCs of goat. (J) CircTGFβ2 was successfully inhibited in MuSCs of goat. (K) The expression of TGFβ2 mRNA was detected by qRT-PCR after circTGFβ2 knockdown. (L) The expression of MyoD, MyoG, and MyHC was detected by qRT-PCR after transfection with pLV-circTGFβ2 and pLV-CiR vector. (M) The expression of MyoD, MyoG, and MyHC was detected by qRT-PCR after transfection with si-1, si-2, and si-3. *P < 0.05, **P < 0.01