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 <i>DENND1A</i> plays a significant role in polycystic ovary syndrome. However, the modulation of <i>DENND1A</i> in GCs remains unclear. Our previous integrated analysis of miRNA-mRNA revealed that the 3'-untranslated region of <i>DENND1A</i> could be a target of chi-miR-324-3p. In this study, we used quantitative reverse transcription polymerase chain reaction (RT-qPCR) to investigate <i>DENND1A</i> 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-<i>DENND1A</i> in GCs. The results revealed an opposite tendency between the expressions of chi-miR-324-3p and <i>DENND1A</i> 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 <i>LHR, Cylin D2</i>, and <i>CDK4</i> showed the same tendency. The dual-fluorescent reporter assay revealed that chi-miR-324-3p directly targeted <i>DENND1A</i>, and the RT-qPCR results revealed that <i>DENND1A</i> expression was inhibited by chi-miR-324-3p. In summary, chi-miR-324-3p inhibited the proliferation of GCs by targeting <i>DENND1A</i>.