METTL3对猪干细胞多能基因表达的调控作用

METTL3 regulates expression of pluripotent genes in porcine pluripotent stem cells

摘要:

m6A是真核生物mRNA中重要的转录后修饰，METTL3作为m6A甲基转移酶复合物中的重要组分，在细胞重编程、胚胎干细胞和诱导多能干细胞的干性维持、胚胎发育等过程中发挥重要作用。为了揭示猪METTL3的表达模式，对不同物种METTL3蛋白序列进行了比对，用RT-PCR检测了METTL3基因在不同猪组织和细胞中的表达情况，并确认了METTL3的细胞核定位。为了研究METTL3对猪干细胞多能基因表达的调控作用，克隆了猪METTL3编码区序列，设计了METTL3干扰片段，并构建了相应的过表达和沉默载体。发现干扰METTL3的表达后，猪多能干细胞出现类似na?ve状态的细胞克隆，NANOG、OCT4和LIN28A表达水平显著升高。在猪多能干细胞培养基中添加m6A甲基化抑制剂环亮氨酸培养细胞48 h后，试验结果与干扰METTL3表达的结果一致。本研究为优化猪多能干细胞的培养体系提供了新的方向和依据。

Abstract:

In post-transcriptional mRNA modification, m6A has been observed in a wide range of eukaryotes. METTL3, as a component of methyltransferase complex for m6A modification, regulates mouse na?ve pluripotency and influences mRNA stability, especially affecting the expression level of the key pluripotent transcription factors. To reveal the expression pattern of the porcine METTL3 gene, we analyzed METTL3 expression level in different porcine tissues, somatic cells, and induced pluripotent stem cells (piPSCs) by RT-PCR. To identify the function of METTL3 for regulation of the expression of porcine pluripotent genes, we cloned a 1 859-bp coding sequence of METTL3 and synthesized a shRNA against METTL3. When knocking down METTL3 expression in piPSCs, the cell type of piPSCs became na?ve-like morphology, alkaline phosphatase activity was increased, and expression level of pluripotent genes NANOG, OCT4 and LIN28A was significantly elevated. In addition, piPSCs cultured in medium containing 10 mmol/L cycloleucine for 48 h exhibited the similar result as that knocked down METTL3.These findings set the stage for optimization of piPS culture condition and further study on the roles of m6A in piPSCs.