miR-194-3 Regulates Proliferation and Apoptosis of Follicular Granulosa Cells by Targeting CHD4 in Zhedong White Geese

The dynamic balance between proliferation and apoptosis of follicular granulosa cells (GCs) is crucial for follicular development in poultry. microRNAs play important roles in ovarian development and follicular function. Previous transcriptome analyses showed that <i>miR-194-3</i> was significantly differentially expressed in the ovaries of Zhedong white geese during the laying and brooding stages. Therefore, the aim of this study was to investigate the regulatory role and molecular mechanism of <i>miR-194-3</i> on the proliferation and apoptosis of follicular GCs in Zhedong white geese. We first screened the target gene <i>CHD4</i> of <i>miR-194-3</i> and constructed the <i>miR-194-3</i> mimic and inhibitor, a small interfering RNA of target gene <i>CHD4</i>. The experimental results showed that the overexpression of <i>miR-194-3</i> significantly down-regulated the mRNA and protein expression of GC proliferation genes (<i>PCNA, CDK-2,</i> and <i>CCND-1</i>), reduced the proportion of EdU-labeled positive cells, blocked cell cycle progression, simultaneously up-regulated the mRNA and protein expression of <i>Caspase-3</i> and <i>Caspase-9</i>, and significantly increased the rate of apoptosis. In contrast, the inhibition of <i>miR-194-3</i> expression promoted the proliferation of goose follicular GCs, accelerated cell cycle progression, and decreased the apoptosis rate. Bioinformatics prediction combined with the results of the dual luciferase reporter assay confirmed that <i>CHD4</i> was a direct target gene of <i>miR-194-3</i>. The knockdown of <i>CHD4</i> expression resulted in the down-regulation of <i>PCNA</i>, <i>CDK-2</i> and <i>CCND-1</i> expression; blockage of cell cycle progression; attenuation of cell proliferation; an up-regulation of <i>Caspase-3</i> and <i>Caspase-9</i> expression and a significant increase in apoptotic cell death. In summary, both <i>miR-194-3</i> overexpression and <i>CHD4</i> knockdown produced similar effects on goose follicular GC proliferation and apoptosis, suggesting that <i>CHD4</i> may partially mediate the regulatory effects of <i>miR-194-3</i>; however, additional targets or pathways cannot be excluded.