鸡METTL14基因调控前脂肪细胞分化的机制研究

m6A RNA methylation plays an important role in the regulation of preadipocyte differentiation. METTL14 is a key m6A methylation regulator, which participates in the differentiation of mouse preadipocytes. To date, the role of METTL14 and how it regulates preadipocyte differentiation remain unknown in chickens. In the current project, we are planning to firstly evaluate the relationship between METTL14 expression, global m6A modification and fat deposition, respectively, by using Western blot and Elisa to detect the expression of METTL14 and global m6A level in abdominal adipose tissue of lean and fat chicken lines and during preadipocyte differentiation. In addition, we will investigate the effects of METTL14 on preadipocyte differentiation, if global m6A level, lipid droplet deposition and adipogenic marker gene expression will be changed by gene over-expression, knockdown and knockout techniques. Finally, we will screen the potential target genes of METTL14 by transcriptome-wide MeRIP-Seq and RNA-Seq assays, and confirm the molecular mechanisms of METTL14 regulating target genes expression and the role of the target genes in adipogenesis by using luciferase reporter assay, chemical treatment and RIP-qPCR. Combining all these approaches, the regulatory mechanism of METTL14 in chicken preadipocyte differentiation will be clarified.

m6A RNA甲基化在前脂肪细胞分化中发挥重要的调控作用。METTL14是一个关键的m6A甲基化调节因子，参与调控小鼠前脂肪细胞分化。鸡METTL14基因对前脂肪细胞分化的影响和调控机制尚不清楚。本项目拟采用Western blot和Elisa技术检测高、低脂系肉鸡腹部脂肪组织中及前脂肪细胞分化过程中METTL14的表达水平及整体m6A水平，明确METTL14表达及整体m6A RNA甲基化与脂肪沉积的关系；随后，采用基因过表达、干扰及敲除技术，观察整体m6A RNA甲基化、脂滴沉积和前脂肪细胞分化标志基因表达的变化，明确鸡METTL14基因对前脂肪细胞分化的影响；最后，利用MeRIP-Seq和RNA-Seq技术筛选METTL14的候选靶基因，之后采用荧光素酶报告系统、化学处理、RIP-qPCR等方法对部分关键靶基因进行验证。通过上述研究，揭示鸡METTL14基因调控前脂肪细胞分化的机制。

选育专门化低脂肉鸡品系是世界范围内肉鸡育种重要的奋斗目标之一。要解决肉鸡体脂过度沉积的问题，需要从根本上了解鸡脂肪组织生长发育的分子遗传学基础。脂肪组织生长发育的细胞学基础是脂肪生成，即脂肪细胞数量的增多和体积的增大。脂肪生成的关键环节之一是前脂肪细胞分化为成熟脂肪细胞。m6A RNA甲基化在前脂肪细胞分化中发挥重要的调控作用。METTL14是一个关键的m6A甲基化调节因子，参与调控小鼠前脂肪细胞分化。然而，METTL14在鸡前脂肪细胞分化中的功能和作用机制尚不清楚。.在本项目中，我们利用RT-qPCR、Western blot和Elisa技术探究了METTL14表达及整体m6A RNA甲基化与脂肪沉积的关系，发现METTL14 mRNA和蛋白表达水平与肉鸡腹脂率呈显著正相关，整体m6A水平与肉鸡腹脂率呈负相关趋势；在鸡前脂肪细胞分化过程中，METTL14 mRNA和蛋白表达水平下降，但整体m6A水平没有明显变化。随后，我们利用过表达技术研究了METTL14在鸡前脂肪细胞分化中的基本功能，发现过表达METTL14能上调整体m6A水平并促进鸡前脂肪细胞分化。在此基础上，我们利用MeRIP-Seq、RNA-Seq、MeRIP-qPCR、RT-qPCR、荧光素酶报告基因等技术研究了METTL14调控鸡前脂肪细胞分化的分子机制，发现BMI1、TMED3、MFAP2是METTL14的靶基因；METTL14以m6A-IGF2BP1/2依赖的方式增加BMI1的mRNA稳定性并上调其表达，同时以m6A-YTHDF2依赖的方式降低TMED3和MFAP2的mRNA稳定性并下调其表达，从而促进鸡前脂肪细胞分化。这些研究成果不但丰富了鸡前脂肪细胞分化的表观遗传调控网络，而且有望应用于低脂肉鸡的分子设计育种中，因此具有良好的应用前景。