The Six-Transmembrane Epithelial Antigen of the Prostate (STEAP) 3 Regulates the Myogenic Differentiation of Yunan Black Pig Muscle Satellite Cells (MuSCs) In Vitro via Iron Homeostasis and the PI3K/AKT Pathway

Abstract

The myogenic differentiation of muscle satellite cells (MuSCs) is an important biological process that plays a key role in the regeneration and repair of skeletal muscles. However, the mechanisms regulating myoblast myogenesis require further investigation. In this study, we found that STEAP3 is involved in myogenic differentiation based on the Yunan black pig MuSCs model in vitro using cell transfection and other methods. Furthermore, the expression of myogenic differentiation marker genes MyoG and MyoD and the number of myotubes formed by the differentiation of cells from the si-STEAP3 treated group were significantly decreased but increased in the STEAP3 overexpression group compared to that in the control group. STEAP3 played a role in iron ion metabolism, affecting myogenic differentiation via the uptake of iron ions and enhancing IRP-IRE homeostasis. STEAP3 also activated the PI3K/AKT pathway, thus promoting myoblast differentiation of Yunan black pig MuSCs. The results of this study showed that STEAP3 overexpression increased intracellular iron ion content and activated the homeostatic IRP-IRE system to regulate intracellular iron ion metabolism.

Keywords: MuSCs myogenic differentiation; PI3K/AKT pathway; STEAP3; iron homeostasis.

Figure 1

Isolation, myogenic differentiation and identification of YuNan black pig MSCs. (A) Identification of porcine primary skeletal muscle satellite cells (Bar: 100 μm). (B) Induced differentiation of porcine primary skeletal muscle satellite cells (Bar: 500 μm, 100 μm). (C) Detection of marker genes for myogenesis. n = 3.

Figure 2

Expression, distribution, and role of STEAP3 in primary muscle satellite cells and muscle tissue. (A) mRNA expression of STEAP family during differentiation. (B) mRNA expression of STEAP3 during differentiation. (C,D) Protein expression of STEAP3 during differentiation. (E) mRNA expression of STEAP3 in skeletal muscle of pigs of different ages. (F,G) Protein expression of STEAP3 in skeletal muscle of pigs of different ages. (H) Distribution of STEAP3 in skeletal muscle of pigs of different ages (Bar: 100 μm). n = 3, * p < 0.05, ** p < 0.01, *** p < 0.01.

Figure 3

Si-STEAP3 on myogenic differentiation. (A) si-STEAP3 sequence screening mRNA detection (1d). (B,C) si-STEAP3 sequence screening protein detection (1d). (D) Interference effect of STEAP3 during differentiation, mRNA verification. (E,F) Interference effect of STEAP3 during differentiation, WB verification. (G,H) Differentiation marker gene detection. (I,J) Myotube MYHC immunofluorescence detection and confluency. The red arrow points to the myotubes formed after myogenic differentiation (Bar: 100 μm). n = 3, * p < 0.05, ** p < 0.01, *** p < 0.01.

Figure 4

Effect of STEAP3 overexpression on myogenic differentiation. (A) plasmid overexpression mRNA detection (1d). (B,C) Plasmid overexpression protein detection (1d). (D) mRNA verification of STEAP3 overexpression effect during differentiation. (E,F) Overexpression effect of STEAP3 during differentiation WB verification. (G,H) Differentiation marker gene detection. (I,J) Myotube MYHC immunofluorescence detection and confluency. The red arrow points to the myotubes formed after myogenic differentiation (Bar: 100 μm). n = 3, * p < 0.05, ** p < 0.01, *** p < 0.01.

Figure 5

STEAP3 regulated iron ions in the regulation of myogenic differentiation via homeostasis. (A) Changes in iron ions during cell differentiation. (B) Changes in iron ions when interfering with STEAP3. (C) Changes in iron ions on overexpression of STEAP3. (D) Changes in iron ions after the addition of DFO and AFC. (E,F) Genetic testing for markers of myogenesis differentiation. (G) Changes in iron metabolism genes when STEAP3 is interfered with. (H) Changes in iron metabolism genes in overexpression of STEAP3. (I,J) Detection of genes of the main pathways of iron metabolism when interfering with and overexpressing STEAP3. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.01.

Figure 6

STEAP3 promotes MUSCs’ myogenic differentiation by regulating the PI3K-AKT signaling pathway. (A,B) Detection of myogenic differentiation genes after the addition of PI3K inhibitors. (C) The iron ion content varies under different conditions. (D,E) Changes in iron homeostasis pathway genes after the addition of PI3K inhibitors. (F) Changes in the PI3K-AKT pathway protein after STEAP3 overexpression. (G) Changes in the PI3K-AKT pathway protein after STEAP3 interference. (H) Changes in intracellular iron pools under different conditions. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.01.

Figure 7

The mechanism of STEAP3 promotes Yunan black pig MuSCs myogenic differentiation.