Abstract: 【Objective】 The purpose of this study was to explore the effect of the expression N⁶-methyladenosine (m⁶A) demethylation enzyme FTO on the differentiation of porcine muscle satellite cells, and compare FTO expression and m⁶A methylation modification levels of pigs with different phenotypes.【Method】 Porcine muscle satellite cells on days 0, 2 and 4 of differentiation were collected, and the protein expressions of FTO and myosin heavy chain (MyHC)and mRNA expression of myogenic differentiation factors (MyoD)and myogenin(MyoG) genes related to muscle cell differentiation were detected by Western blotting and Real-time quantitative PCR.The expression of MyHC, a marker of myocyte differentiation, was detected by immunofluorescence assay.Dot blotting was used to detect the methylation level of m⁶A.Overexpressed vector (OE-FTO), blank control (NC), interference vector (siRNA-FTO) and negative control (siRNA-NC) of FTO gene were transfected into porcine muscle satellite cells and induced differentiation, respectively.The expression of FTO and myocyte differentiation related genes and the methylation modification level of m⁶A were detected.Immunofluorescence was used to detect the expression of MyHC and the formation of muscle tubes.At the same time, Western blotting and Dot blotting were used to detect FTO protein expression and m⁶A methylation modification level in different tissues of Large White pigs and Ningxiang pigs.【Result】 Compared with day 0, the expression of FTO and MyHC protein were extremely significantly increased on days 2 and 4 (P<0.01), and the mRNA expression of FTO, MyoD and MyoG genes were significantly or extremely significantly increased (P<0.05 or P<0.01).The methylation modification level of m⁶A was significantly decreased on days 4 (P<0.05).Compared with NC group, the expression of FTO protein in OE-FTO group was extremely significantly increased (P<0.01), and the mRNA expression of FTO and MyHC genes in OE-FTO group were extremely significantly increased on days 0, 1 and 2 of induction differentiation (P<0.01), and the mRNA expression of MyoD gene was extremely significantly decreased (P<0.01).On days 1, 2, 3 and 4, the methylation modification level of m⁶A in OE-FTO group was significantly or extremely significantly decreased (P<0.05 or P<0.01).Compared with siRNA-NC group, the expression of FTO protein in siRNA-FTO group was significantly decreased (P<0.05), and the mRNA expression of FTO and MyHC genes in siRNA-FTO group were extremely significantly or significantly decreased on days 0, 1, 2 and 3 of induction differentiation (P<0.01 or P<0.05).The methylation level of m⁶A was significantly or extremely significantly increased (P<0.05 or P<0.01);The mRNA expression of MyoG gene in siRNA-FTO group was significantly decreased on days 0, 2 and 3 of induction differentiation (P<0.05).The expression of FTO protein in longissimus dorsi muscle of Large White pigs was significantly higher than that of Ningxiang pigs (P<0.05), and the methylation level of m⁶A was significantly extremely lower than that of Ningxiang pigs (P<0.01).【Conclusion】 The expression of FTO had a significant effect on the differentiation of porcine muscle satellite cells, and the methylation level of m⁶A was negatively correlated with the differentiation degree of porcine muscle satellite cells.Interference with FTO inhibited the differentiation of porcine muscle satellite cells and increased the methylation level of m⁶A.Overexpression of FTO could promote the differentiation of porcine muscle satellite cells and reduce the methylation level of m⁶A.This results provided reference for further exploring the regulatory mechanism of FTO in muscle differentiation.

Key words: N⁶-methyladenosine (m⁶A); FTO; porcine muscle satellite cells; induction differentiation