NTN1 Affects Porcine Intramuscular Fat Content by Affecting the Expression of Myogenic Regulatory Factors

SIMPLE SUMMARY: Intramuscular fat (IMF) is a key meat quality trait in the pork industry. In this study, we validated the effect of the copy number of Netrin-1 (NTN1-CNV) on Netrin-1 (NTN1) protein expression and explored the possible affective mechanism of NTN1 on IMF. The results indicated that NTN1-CNV may affect the expression of NTN1 protein by its gene dose, and the expression of NTN1 may affect the proliferation and differentiation of muscle cells by the AMP-activated protein kinase (AMPK) pathway and finally influence the IMF content. ABSTRACT: Intramuscular fat (IMF) content is an important economic trait for pork quality. Our previous results regarding the genome-wide association between IMF content and copy number variations (CNVs) indicated that the CNV within Netrin-1(NTN1-CNV) was significantly associated with IMF. In order to validate the effect of NTN1-CNV, we detected the Netrin-1 (NTN1) gene dose and protein expression content in the longissimus dorsi of different IMF content pigs using Western blotting and investigated the expression of NTN1 RNA in different tissues using real-time quantitative polymerase chain reaction (qPCR). The knock-down of the NTN1 gene in C2C12 and 3T3-L1 cells and over-expression in C2C12 cells during the proliferation and differentiation stage were also investigated to explore the possible pathway of action of NTN1. The results showed that in individuals with IMF content differences, the gene dose of NTN1 and the expression of NTN1 protein were also significantly different, which indicated that NTN1-CNV may directly affect IMF by its coding protein. NTN1 had the highest expression in pig longissimus dorsi and backfat tissues, which indicates that NTN1 may play an important role in muscle and fat tissues. The in vitro validation assay indicated that NTN1 silencing could promote the proliferation and inhibit the differentiation of C2C12 cells, with no effect on 3T3-L1 cells. Additionally, NTN1 over-expression could inhibit the proliferation and promote the differentiation of C2C12 cells. Combined with previous research, we conclude that NTN1-CNV may affect IMF by its gene dose, and the expression of NTN1 may affect the proliferation and differentiation of muscle cells by the AMP-activated protein kinase (AMPK) pathway and finally influence the IMF.