m^(6)A甲基化修饰相关酶基因在牛脂肪生成中的表达分析

Expression Analyses of m^(6)A Methylase Genes in Bovine Adipogenesis

探究参与m^(6)A修饰的相关酶基因包括m^(6)A甲基转移酶METTL3、METTL14、WTAP和m^(6)A去甲基化酶FTO、ALKBH5,在牛不同组织以及前体脂肪细胞增殖和分化过程中的表达水平,为阐明m^(6)A修饰在脂生成中的功能研究提供参考。使用实时荧光定量PCR技术检测基因在新生和成年秦川肉牛不同组织,以及在前体脂肪细胞中的表达;采用CCK-8和油红O染色检测前体脂肪细胞的生长曲线及其成脂分化中的脂滴形成情况。5个基因均在骨骼肌中低表达,其余组织中高表达,而在成年牛脂肪组织的表达水平均显著高于新生牛(P<0.05)。分离的前体脂肪细胞具有良好的生长状态且可正常成脂分化。在牛前体脂肪细胞增殖期,METTL3、METTL14和WTAP的表达在前期下降,之后有所上升但仍维持较低的表达水平。FTO的表达呈缓慢上升趋势。ALKBH5在增殖前期上升,中期下降,在第4天后不断增加(P<0.01)。在前体脂肪细胞分化过程中,METTL3、METTL14和FTO的表达模式基本一致,在分化前期下降,中期高表达,随后降低。而WTAP和ALKBH5的表达随分化进行逐渐增加。结果表明m^(6)A修饰对秦川牛脂肪发育和沉积具有潜在的重要作用,且m^(6)A甲基化酶可能在体外调控牛前体脂肪细胞的脂生成。

The aim of this study is to investigate the mRNA expressions of relevant genes,including METTL3,METTL14,WTAP for m^(6)A methyltransferase,and FTO and ALKBH5 for m^(6)A demethylaes,in bovine various tissues and in the proliferation and differentiation of preadipocytes,aiming to provide a reference for clarifying the role and mechanism of m^(6)A modification in adipogenesis.Real-time quantitative PCR(RT-qPCR)was used to detect the mRNA expressions of m^(6)A methylases in the various tissues of newborn and adult Qingchuan beef cattle.Then,CCK-8 treatment and oil-red O staining were adapted to detect the growth curves of the preadipocytes cells and lipid droplet formation during adipogenic differentiation.The 5 genes expressed lowly in skeletal muscles,and highly in rest tissues,while the mRNA expressions of them in the adipose tissue of adult cattle was significantly higher than that of newborn cattle(P < 0.05).The isolated preadipocytes had a good growth state and adipogenic differentiation was accordance with the general pattern.The expressions of METTL3,METTL14 and WTAP decreased in the early stage of bovine preadipocytes proliferation,then increased and remained at low level.The expression of FTO showed a slow upward trend.The temporal expression of ALKBH5 increased in the early stage,decreased in the metaphase,and increased continuously after the day 4(P < 0.01).The expression patterns of METTL3,METTL14 and FTO were similar in the adipogenic differentiation of bovine preadipocytes,which decreased in the early stage of differentiation,and highly expressed in the metaphase,then decreased.The expressions of WTAP and ALKBH5 increased with the progress of differentiation.Our results indicate that m^(6)A modification plays a potentially important role in fat development and deposition of Qinchuan cattle,and m^(6)A methylases may regulate the lipogenesis of bovine preadipocytes in vitro.