MicroRNA-27a-3p targeting Vangl1 and Vangl2 inhibits cell proliferation in mouse granulosa cells

Abstract

BackgroundMammalian folliculogenesis is the complex process through which primordial follicles develop into preovulatory follicles. The chief function of ovarian follicle granulosa cells is to play a vital role in the growth, development and atresia of ovarian follicles via gap junctions. Increasing evidence suggests that microRNAs (miRNAs) are essential regulators of granulosa cell apoptosis or proliferation. MethodsThe expression level of miR-27a-3p, myogenic differentiation (MyoD), Vangl1 and Vangl2 was investigated by Real-time quantitative PCR (RT-qPCR) and Western blot. Luciferase reporter assay, bioinformatics analysis and ChIP-PCR was used to detect the binding sites between miR-27a-3p, transcription factor and target genes. KEGG pathway analyses were performed to reveal the predicted targets of miR-27a-3p. Ethynyl deoxyuridine (EdU) proliferation assay was used to measure cell proliferation. ResultsTo explore the underlying mechanisms of the miR-27a-3p function in the development of mouse granulosa cells (mGCs), we screened for the target genes of miR-27a-3p, confirmed its interaction with Vangl1 and Vangl2 and elucidated their roles in mGCs. MiR-27a-3p inhibited the proliferation of mGCs, whereas target genes Vangl1 and Vangl2 had the opposite effect. In addition, the transcription factor MYOD bound to and activated the promoter of miR-27a-3p. MiR-27a-3p suppressed Vangl1 and Vangl2 expression by targeting their 3′-untranslated region (3′-UTR). Furthermore, Vangl1 and Vangl2 suppressed the Wnt pathway by reducing the expression of β-catenin and B-cell lymphoma/leukemia-2 (Bcl-2). ConclusionThese findings indicate a pro-survival mechanism of the MyoD/miR-27a-3p/Vangl1/Vangl2 axis for granulosa cell proliferation and suggest a novel target for the improvement of female fertility.