Regulates Proliferation and Apoptosis of Follicular Granulosa Cells by Targeting

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 miR-194-3 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 miR-194-3 on the proliferation and apoptosis of follicular GCs in Zhedong white geese. We first screened the target gene CHD4 of miR-194-3 and constructed the miR-194-3 mimic and inhibitor, a small interfering RNA of target gene CHD4. The experimental results showed that the overexpression of miR-194-3 significantly down-regulated the mRNA and protein expression of GC proliferation genes (PCNA, CDK-2, and CCND-1), reduced the proportion of EdU-labeled positive cells, blocked cell cycle progression, simultaneously up-regulated the mRNA and protein expression of Caspase-3 and Caspase-9, and significantly increased the rate of apoptosis. In contrast, the inhibition of miR-194-3 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 CHD4 was a direct target gene of miR-194-3. The knockdown of CHD4 expression resulted in the down-regulation of PCNA, CDK-2 and CCND-1 expression; blockage of cell cycle progression; attenuation of cell proliferation; an up-regulation of Caspase-3 and Caspase-9 expression and a significant increase in apoptotic cell death. In summary, both miR-194-3 overexpression and CHD4 knockdown produced similar effects on goose follicular GC proliferation and apoptosis, suggesting that CHD4 may partially mediate the regulatory effects of miR-194-3; however, additional targets or pathways cannot be excluded.