Abstract:

This study aimed to investigate the regulatory impact of TMEM182 on the proliferation and differentiation of porcine skeletal muscle satellite cells. This study utilized 3 each of healthy castrated boars, Mashen pigs and Large White pigs, housed under the same conditions for 180 days of age, were selected for collection of each tissue; one-week-old piglets were selected for isolation of porcine skeletal muscle satellite cells; qRT-PCR was used to investigate the expression profile of TMEM182 in different pig tissues, compare expression patterns in muscle tissues across various pig species, and analyze the temporal expression changes in skeletal muscle satellite cells during different stages of differentiation. By overexpressing TMEM182 (OE-TMEM182) and interfering with TMEM182 (si-TMEM182) in satellite cells, we utilized qRT-PCR to analyze the expression changes of proliferation marker genes and conducted EdU experiments to observe changes in the number of EdU-positive cells. Following the induction of myogenic differentiation in porcine skeletal muscle satellite cells, various techniques including qRT-PCR, Western blot, and immunofluorescence staining techniques were utilized to examine alterations in the mRNA and protein expression levels of genes associated with myogenic differentiation, as well as their impact on myotube fusion. The study results showed that TMEM182 had high expression levels in muscle tissues but was nearly absent in other tissues. In comparison to Mashen pigs, TMEM182 was notably more expressed in Large White pigs tissues (P < 0.05). As satellite cells differentiation progressed, at the 4th and 5th days of differentiation, there was an increase in TMEM182 expression. The expression level was the lowest on the 0th day of differentiation. However, at the 6th day of differentiation, there was a decrease in TMEM182 expression. Conversely, at the 7th day of differentiation, TMEM182 expression was at its peak(P < 0.05). The results of the intercalated proteins showed that the expression of CDH15, TMEM25, HS3ST1, and SEC11A was highly significantly reduced in the OE-TMEM182 group (P < 0.01), the expression of VWC2L was significantly reduced (P < 0.05).During the proliferation phase, overexpression of TMEM182 significantly down-regulated the expression of CDK1, PCNA, and Ki67 (P < 0.01), together with the number of EdU-positive cells was markedly decreased (P < 0.01). Conversely, interfering with TMEM182 resulted in an highly upregulation the expressions of CDK4, CDK1 (P < 0.01) and significantly upregulated the expression of Ki67 (P < 0.05), along with an increase in EdU-positive cells (P < 0.01). Furthermore, during myogenic differentiation, overexpression of TMEM182 led to a highly significant reduction in the expression levels of MyoG, MyHC (P < 0.01) and the expression level of MyoD was significantly decreased (P < 0.05). After interfering with TMEM182, the expression of MyoD, MyoG and MyHC were highly significantly increased (P < 0.01) and the expression of Myf5 was significantly increased (P < 0.05). At the same time, TMEM182 might regulate the proliferation and differentiation of porcine satellite cells by inhibiting PI3K-Akt signaling pathway. The study results indicate the crucial role of TMEM182 as a negative regulator in the proliferation and differentiation of porcine skeletal muscle satellite cells. This gene could be a key candidate gene influencing the growth and development of porcine skeletal muscle.

Key words: TMEM182, muscle-specific, skeletal muscle satellite cells, proliferation, myoblast differentiation