chi-miR-324-3p Regulates Goat Granulosa Cell Proliferation by Targeting DENND1A

Abstract

Granulosa cell (GC) proliferation provides essential conditions for ovulation in animals. A previous study showed that DENND1A plays a significant role in polycystic ovary syndrome. However, the modulation of DENND1A in GCs remains unclear. Our previous integrated analysis of miRNA–mRNA revealed that the 3'-untranslated region of DENND1A could be a target of chi-miR-324-3p. In this study, we used quantitative reverse transcription polymerase chain reaction (RT-qPCR) to investigate DENND1A expression in ovarian tissues of high- and low-yielding goats. Furthermore, dual-fluorescent reporter vector experiments, Cell Counting Kit-8 (CCK-8) assay, and RT-qPCR were used to elucidate the regulatory pathway of chi-miR-324-3p-DENND1A in GCs. The results revealed an opposite tendency between the expressions of chi-miR-324-3p and DENND1A in the ovaries of high- and low-yielding goats. The CCK-8 assay indicated that chi-miR-324-3p overexpression significantly suppressed GC proliferation, whereas chi-miR-324-3p inhibition promoted GC proliferation. In addition, the expressions of GC proliferation markers LHR, Cylin D2, and CDK4 showed the same tendency. The dual-fluorescent reporter assay revealed that chi-miR-324-3p directly targeted DENND1A, and the RT-qPCR results revealed that DENND1A expression was inhibited by chi-miR-324-3p. In summary, chi-miR-324-3p inhibited the proliferation of GCs by targeting DENND1A.