ABSTRACT

1. MicroRNAs (miRNAs) play a vital role in the proliferation, differentiation, and apoptosis of myoblasts. However, the effect of miR-7 on the apoptosis of chicken primary myoblasts (CPMs) and its mechanism is still unclear.

2. In this study, the expression of apoptosis marker genes (RAF1, Caspase3, Caspase9, Cytc, Fas) in CPMs was significantly increased after transfection of miR-7 mimic. The expression of the apoptosis marker genes in CPMs was significantly reduced after transfection with miR-7 inhibitor. Flow cytometry showed that the late apoptosis rate of the mimic group was significantly higher than the negative control (NC). The viable cells of the mimic group were significantly lower than the NC. In contrast, inhibition of miR-7 had the opposite effect.

3. The dual-luciferase assay showed that the KLF4 was a target gene of miR-7. The rescue experiment showed that the KLF4 gene could attenuate the effect of miR-7 on the expression of apoptosis marker genes in CPMs.

4. Determination of the function the KLF4 gene showed that the expression of the apoptosis marker genes in CPMs decreased significantly compared with the NC after its overexpression. Inhibition of KLF4 gene had the opposite effect. Flow cytometry showed that overexpression of the KLF4 gene inhibited early apoptosis of myoblasts (P ≤ 0.01), while interference with the KLF4 gene could promote early apoptosis of myoblasts (P ≤ 0.001).

5. The results demonstrated, for the first time, that miR-7 promotes apoptosis in chicken primary myoblasts by regulating the expression of the KLF4 gene.

摘要

1. MicroRNAs (miRNAs) 在成肌细胞的增殖、分化和凋亡中发挥着至关重要的作用。然而，miR-7对鸡原代成肌细胞（CPMs）凋亡的影响及其机制尚不清楚。

2、本研究中，转染miR-7 mimic后CPMs细胞凋亡标志基因（RAF1、Caspase3、Caspase9、Cytc、Fas）的表达明显增加。转染miR-7抑制剂后，CPMs中凋亡标记基因的表达显着降低。流式细胞仪显示，模拟组的晚期凋亡率明显高于阴性对照（NC）。模拟组的活细胞显着低于NC。相反，抑制miR-7具有相反的效果。

3.双荧光素酶检测显示KLF4是miR-7的靶基因。拯救实验表明，KLF4基因可以减弱miR-7对CPMs细胞凋亡标记基因表达的影响。

4、功能测定KLF4基因过表达后，CPMs中凋亡标记基因的表达较NC显着降低。KLF4基因的抑制作用相反。流式细胞术显示KLF4基因过表达抑制成肌细胞早期凋亡（P≤0.01），而干扰KLF4基因可促进成肌细胞早期凋亡（P≤0.001）。

5.结果首次证明miR-7通过调节KLF4基因的表达促进鸡原代成肌细胞凋亡。