miRNA-200a通过靶向ZEB1参与调节鸡胸肌细胞增殖分化

ZEB1在细胞增殖分化中发挥关键作用,然而关于ZEB1在鸡胸肌细胞增殖分化过程中的功能及其与miRNA互作的研究极少。为探索miRNA如何通过靶向ZEB1参与调节鸡胸肌细胞增殖分化,实验检测了ZEB1在55周龄和20周龄鸡胸肌组织中的表达,使用Target Scan及miRDB在线软件预测鸡ZEB1基因的靶向miRNA,构建ZEB1野生型、突变型双荧光报告载体,并在DF1细胞中验证了ZEB1的靶向miRNA,双荧光素酶报告实验结果说明miR-200a通过特异性结合ZEB1 3'非编码区种子序列直接靶向并抑制ZEB1基因的表达。结果表明:由于miR-200a在55周龄鸡胸肌表达下调,对ZEB1的抑制作用减弱,导致ZEB1在55周龄固始鸡胸肌组织表达较20周龄显著升高(P0.01)。本研究首次在鸡上证明miR-200a是ZEB1的靶向miRNA,且miR-200a可能通过靶向ZEB1参与调节鸡胸肌细胞的增殖分化,为深入理解miRNAs在家禽及其他鸟类中的分子调节机制提供了基础与依据。

Study on the Role of gga(Gallus Gallus)-miRNA-200a in Regulating Cell Differentiation and Proliferation of Chicken Breast Muscle by Targeting ZEB1

A lot of studies have proved that Zinc finger E-box binding homeobox 1, ZEB1 played a major role in cell proliferation, cell survival and cell differentiation. However, the regulation of ZEB1 expression by microRNAs(miRNAs) in chicken breast muscle still remains unknown. This experiment examined the expression of ZEB1 in chicken breast tissues at 55 weeks and 20 weeks of age. In this study, Target Scan and miRDB software were used to predict the target miRNA of chicken ZEB1 gene, and ZEB1 wild-type and mutant double fluorescent reporter vector were constructed, the relationship between the potential miRNA and ZEB1 was verified using dual luciferase reporter assay in chicken DF1 cells. Dual-luciferase reporter assays results demonstrated that the expression of luciferase reporter gene linked with part sequence of the 3′UTR of chicken ZEB1 gene was down-regulated by the overexpression of gga-miR-200a in the DF1 cells, and the down-regulation behavior was abolished when the gga-miR-200a binding site in 3′UTR of ZEB1 was mutated, indicating that gga-miR-200a can suppress the expression level of its target gene ZEB1. Therefore, we concluded that gga-miR-200a might be involved in the cell differentiation and proliferation of chicken breast muscle through binding with ZEB1 3'UTR. These findings will contribute to further understanding of the regulatory mechanisms of miRNAs in chickens.