RASGRP1 targeted by H3K27me3 regulates myoblast proliferation and differentiation in mice and pigs

Abstract

Skeletal muscle is not only the largest organ in the body that is responsible for locomotion and exercise but also crucial for maintaining the body's energy metabolism and endocrine secretion. The trimethylation of histone H3 lysine 27 (H3K27me3) is one of the most important histone modifications that participates in muscle development regulation by repressing the transcription of genes. Previous studies indicate that the RASGRP1 gene is regulated by H3K27me3 in embryonic muscle development in pigs, but its function and regulatory role in myogenesis are still unclear. In this study, we verify the crucial role of H3K27me3 in RASGRP1 regulation. The gain/loss function of RASGRP1 in myogenesis regulation is performed using mouse myoblast C2C12 cells and primarily isolated porcine skeletal muscle satellite cells (PSCs). The results of qPCR, western blot analysis, EdU staining, CCK-8 assay and immunofluorescence staining show that overexpression of RASGRP1 promotes cell proliferation and differentiation in both skeletal muscle cell models, while knockdown of RASGRP1 leads to the opposite results. These findings indicate that RASGRP1 plays an important regulatory role in myogenesis in both mice and pigs.

Keywords: H3K27me3; RASGRP1; cell proliferation; myoblasts; skeletal muscle.

Figure 1

RASGRP1 is targeted by H3K27me3 and upregulated in the longissimus dorsi muscle of Duroc pig embryos at gestation days 33 (E33), 65 (E65), and 90 (E90) (A) The enrichment of H3K27me3 in the RASGRP1 promoter at E33, E65, and E90. (B) The expression level of RASGRP1 in pig skeletal muscle at E33, E65, E90 and the adult period. β-Actin was used as a control. *P<0.05, **P<0.01.

Figure 2

RASGRP1 overexpression promotes the proliferation of C2C12 cells (A) The relative expression levels of RASGRP1 and proliferation marker genes after RASGRP1 overexpression. (B) Detection of C2C12 cell viability by CCK-8 assay at 18 h, 24 h and 30 h after RASGRP1 overexpression. (C) The relative protein levels of the Ki67 and CDK2 genes after RASGRP1 overexpression. (D) EdU staining after RASGRP1 overexpression in C2C12 cells. Scale bar: 100 μm. (E) Detection of the number of S-phase cells by flow cytometry after RASGRP1 overexpression. *P<0.05, **P<0.01.

Figure 3

RASGRP1 knockdown inhibits the proliferation of C2C12 cells (A) The relative expression levels of the RASGRP1 gene and proliferation marker genes after RASGRP1 knockdown. (B) Detection of C2C12 cell viability by CCK-8 assay at 18 h and 24 h after RASGRP1 knockdown. (C) The relative protein expression levels of the Ki67 and CDK2 genes after RASGRP1 knockdown. (D) EdU staining after RASGRP1 knockdown in C2C12 cells. Scale bar: 100 μm. *P<0.05, **P<0.01.

Figure 4

RASGRP1 overexpression promotes the differentiation of C2C12 cells (A) Expression levels of differentiation marker genes during differentiation. (B) The relative expression level of RASGRP1 was increased significantly and then decreased during differentiation. (C) The relative expression levels of RASGRP1 and differentiation marker genes after RASGRP1 overexpression. (D) The relative protein expression of MyHC, MyoD and MyoG after RASGRP1 overexpression. (E) MyHC immunofluorescence assay after RASGRP1 overexpression in C2C12 cells. Scale bar: 50 μm. *P<0.05, **P <0.01.

Figure 5

RASGRP1 knockdown inhibits the differentiation of C2C12 cells (A) The relative expressions of RASGRP1 and differentiation marker genes after RASGRP1 knockdown. (B) The relative protein expression levels of MyHC, MyoD and MyoG after RASGRP1 knockdown. (C) MyHC immunofluorescence assay after RASGRP1 knockdown in C2C12 cells. Scale bar: 50 μm. *P<0.05, **P<0.01.

Figure 6

RASGRP1 promotes the proliferation and differentiation of PSCs (A) The relative expression levels of RASGRP1 and proliferation marker genes after RASGRP1 overexpression. (B) Detection of PSC viability by CCK-8 assay at 6 h, 48 h and 72 h after RASGRP1 overexpression. (C) EdU staining after RASGRP1 overexpression in PSCs. Scale bar: 100 μm. (D) The relative expression levels of RASGRP1 and proliferation marker genes after RASGRP1 knockdown. (E) Detection of PSC viability at 6 h, 48 h and 72 h after RASGRP1 knockdown. (F) EdU staining after RASGRP1 knockdown in PSCs. Scale bar: 100 μm. (G) The expression of RASGRP1 at different differentiation stages of PSCs. (H) MyHC immunofluorescence assay after RASGRP1 overexpression in PSCs. Scale bar: 100 μm. (I) MyHC immunofluorescence assay after RASGRP1 knockdown in PSCs. Scale bar: 100 μm. *P<0.05, **P<0.01.