RNA Sequencing Reveals the Regulation of Betaine on Chicken Myogenesis

Abstract

Betaine is trimethylglycine and a universal methyl donor which could provide methyl and glycine for cells and animals. As a new star in epigenetics, N6-Methyladenosine has been reported to regulate multiple biological activities, but the regulatory mechanism of betaine on N6-Methyladenosine as well as myogenesis was little studied. In this study, we treated chicken primary myoblast cells with different concentrations of betaine (0, 10, 25, and 50 mmol/L) and found that myoblast cell proliferation was inhibited, although the cell cycle was promoted in the S phase by betaine, where the myotube area was increased as well as the differentiation marker genes MyoD, MyoG, MyHC, Myomarker, and Ckm. RNA sequencing obtained a total of 61 differentially expressed genes (DEGs); DEGs caused by 50 mmol/L betaine were mainly enriched in the regulation of skeletal muscle tissue regeneration and some amino acid metabolic processes. The gene expression pattern trends of all DEGs were mainly clustered into 2 profiles, with the increase in betaine concentration, the gene expression pattern either increased or decreased continuously. Overall, a low concentration betaine can increase the N6-Methyladenosine modification level and myotube area but depresses myoblast cell proliferation in vitro.

Keywords: N6-Methyladenosine; betaine; chicken; mRNA-Seq; myogenesis.

Figure 1

Betaine inhibits myoblast cells proliferation. (A) RNA dot blot and methylene blue results after being treated with 0, 10, 25, 50 mmol/L betaine for 24 h. (B) The relative gray value of RNA dot blot. (C) Cell cycle analysis of different cell stages after 24 h betaine treatment. (D) The statistical results of CCK-8 assay after 24 h, 36 h, and 48 h betaine treatment. (E) The EdU positive cell rate (F) and EdU staining of myoblast cells being treated with betaine. (mM refers to mmol/L; * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 2

Betaine promotes myotube formation. (A) RNA dot blot and methylene blue results during myoblast differentiation. (B) The relative gray value of RNA dot blot. (C) qRT-PCR results for MyoD, MyoG, MyHC, Myomarker, and Ckm in myoblast cells after being treated with 0, 10, 25, 50 mmol/L betaine for 24 h. (D–F) Myotube area (%) analysis (D), fusion index € of MF20 immunofluorescence staining (F) after being treated with 0, 10, 25, 50 mmol/L betaine for 24 h at DM3. (mM refers to mmol/L; * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 3

Overview of mRNA sequencing in betaine-treated myoblast cells. (A) Venn diagram shows common and unique DEGs between groups. (B) The number of differentially up- and down-expressed genes in each group. (C) GO functional enrichment analysis of DEGs in Bet_0 vs. Bet_50. (D) KEGG pathway analysis of DEGs in Bet_0 vs. Bet_50. (E) Heatmap for all 61 DEGs.

Figure 4

Gene expression pattern analysis of all DEGs. (A) General chart of all gene expression trend profiles. (B) Gene expression trend profiles are ordered based on the p-value significance of the number of genes assigned versus expected. (C, D) the FPKM value (C) and qRT-PCR validation (D) of eight DEGs in profile 0 and 19. (mM refers to mmol/L; * p < 0.05; ** p < 0.01; *** p < 0.001.)