m6A去甲基化酶FTO对猪肌卫星细胞分化的影响

doi:10.16431/j.cnki.1671-7236.2023.07.018

摘要/Abstract

摘要： 【目的】探究N⁶-甲基腺苷（m⁶A）去甲基化酶FTO表达水平对猪肌卫星细胞分化的影响，并比较不同表型猪FTO表达和m⁶A甲基化修饰水平。【方法】收集分化第0、2和4天的猪肌卫星细胞，用Western blotting和实时荧光定量PCR分别检测FTO和肌球蛋白重链（MyHC）蛋白表达、肌分化因子（MyoD）和肌细胞生成素（MyoG）的mRNA表达水平；利用免疫荧光法检测肌卫星细胞分化标志基因MyHC表达情况；采用Dot blotting检测m⁶A甲基化修饰水平。将过表达载体（OE-FTO）、空白对照（NC）和FTO基因干扰载体（siRNA-FTO）、阴性对照（siRNA-NC）分别转染猪肌卫星细胞并诱导分化，检测FTO、肌细胞分化相关基因表达情况以及m⁶A甲基化修饰水平，利用免疫荧光法检测MyHC表达以及肌管形成情况；利用Western blotting和Dot blotting分别检测大白猪、宁乡猪不同组织中FTO蛋白表达情况以及m⁶A甲基化修饰水平。【结果】在猪肌卫星细胞诱导分化过程中，与诱导分化第0天相比，第2和4天时FTO、MyHC蛋白表达水平极显著升高（P<0.01），FTO、MyoD和MyoG基因mRNA表达水平显著或极显著升高（P<0.05；P<0.01）；第4天时m⁶A甲基化修饰水平显著下降（P<0.05）。与NC组相比，OE-FTO组FTO蛋白表达量极显著升高（P<0.01），在诱导分化的第0、1和2天，OE-FTO组FTO和MyHC基因mRNA表达水平均极显著升高（P<0.01），MyoD基因mRNA表达水平极显著下降（P<0.01）；在诱导分化的第1、2、3和4天，OE-FTO组m⁶A甲基化修饰水平显著或极显著下降（P<0.05；P<0.01）。与siRNA-NC组相比，siRNA-FTO组FTO蛋白表达水平显著降低（P<0.05），在诱导分化第0、1、2和3天，siRNA-FTO组FTO、MyHC基因mRNA表达水平极显著或显著降低（P<0.01；P<0.05），m⁶A甲基化修饰水平显著或极显著升高（P<0.05；P<0.01）；在诱导分化第0、2和3天，siRNA-FTO组MyoG基因mRNA表达水平显著降低（P<0.05）。大白猪背最长肌中FTO蛋白表达水平显著高于宁乡猪（P<0.05），m⁶A甲基化修饰水平极显著低于宁乡猪（P<0.01）。【结论】FTO表达对猪肌卫星细胞分化过程有显著影响，m⁶A甲基化修饰水平与猪肌卫星细胞分化程度呈负相关。干扰FTO会抑制猪肌卫星细胞分化，提高m⁶A甲基化修饰水平；过表达FTO可以促进猪肌卫星细胞分化，降低m⁶A甲基化修饰水平。本研究结果为进一步探究FTO在肌肉分化中调控机制提供参考。

关键词: N⁶-甲基腺苷(m⁶A); FTO; 猪肌卫星细胞; 诱导分化

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

中图分类号:

Q291

任祖凤, 顾浩, 胡康洪, 毕延震. m⁶A去甲基化酶FTO对猪肌卫星细胞分化的影响[J]. 中国畜牧兽医, 2023, 50(7): 2777-2788.

REN Zufeng, GU Hao, HU Kanghong, BI Yanzhen. Effect of m⁶A Demethylase Enzyme FTO on Differentiation of Porcine Muscle Satellite Cells[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2777-2788.

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m⁶A去甲基化酶FTO对猪肌卫星细胞分化的影响

Effect of m⁶A Demethylase Enzyme FTO on Differentiation of Porcine Muscle Satellite Cells

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