Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan 430064, China.
Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan 430064, China.
Biochim Biophys Acta Gene Regul Mech. 2023 Mar;1866(1):194885. doi: 10.1016/j.bbagrm.2022.194885. Epub 2022 Oct 23.
Mammalian 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.
The 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.
To 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).
These 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.
哺乳动物卵泡发生是原始卵泡发育为排卵前卵泡的复杂过程。卵巢卵泡颗粒细胞的主要功能是通过缝隙连接在卵泡的生长、发育和闭锁中发挥重要作用。越来越多的证据表明，微小 RNA（miRNA）是颗粒细胞凋亡或增殖的重要调节因子。
采用实时定量 PCR（RT-qPCR）和 Western blot 检测 miR-27a-3p、成肌分化（MyoD）、Vangl1 和 Vangl2 的表达水平。荧光素酶报告基因检测、生物信息学分析和 ChIP-PCR 用于检测 miR-27a-3p、转录因子和靶基因之间的结合位点。KEGG 通路分析用于预测 miR-27a-3p 的靶基因。采用 Ethynyl deoxyuridine（EdU）增殖实验检测细胞增殖。
为了探讨 miR-27a-3p 在小鼠颗粒细胞（mGCs）发育中的作用机制，我们筛选了 miR-27a-3p 的靶基因，证实了它与 Vangl1 和 Vangl2 的相互作用，并阐明了它们在 mGCs 中的作用。miR-27a-3p 抑制 mGCs 的增殖，而靶基因 Vangl1 和 Vangl2 则有相反的作用。此外，转录因子 MYOD 结合并激活了 miR-27a-3p 的启动子。miR-27a-3p 通过靶向其 3'-非翻译区（3'-UTR）抑制 Vangl1 和 Vangl2 的表达。此外，Vangl1 和 Vangl2 通过降低β-catenin 和 B 细胞淋巴瘤/白血病-2（Bcl-2）的表达来抑制 Wnt 通路。
这些发现表明 MyoD/miR-27a-3p/Vangl1/Vangl2 轴对颗粒细胞增殖具有促生存作用，并为提高女性生育能力提供了新的靶点。
