National Engineering Research Center of Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, and Key Lab of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture, South China Agricultural University, Guangzhou 510642, China.
Int J Mol Sci. 2020 Jul 23;21(15):5217. doi: 10.3390/ijms21155217.
Circular RNAs (circRNAs) are thought to play essential roles in multiple biological processes, including apoptosis, an important process in antral follicle atresia. We aimed to investigate the potential involvement of circRNAs in granulosa cell apoptosis and thus antral follicle atresia. CircRNA expression profiles were generated from porcine granulosa cells isolated from healthy antral (HA) and atretic antral (AA) follicles. Over 9632 circRNAs were identified, of which 62 circRNAs were differentially expressed (DE-circRNAs). Back-splicing, RNase R resistance, and stability of DE-circRNAs were validated, and miRNA binding sites and related target genes were predicted. Two exonic circRNAs with low false discovery rate (FDR) high fold change, miRNA binding sites, and relevant biological functions-circ_CBFA2T2 and circ_KIF16B-were selected for further characterization. qRT-PCR and linear regression analysis confirmed expression and correlation of the targeted genes-the antioxidant gene (potential target of circ_CBFA2T2) and the apoptotic gene (potential target of circ_KIF16B). Increased mRNA content of in granulosa cells of AA follicles was further confirmed by strong immunostaining of both p53 and its downstream target pleckstrin homology like domain family a member 3 (PHLDA3) in AA follicles compared to negligible staining in granulosa cells of HA follicles. Therefore, we concluded that aberrantly expressed circRNAs presumably play a potential role in antral follicular atresia.
环状 RNA（circRNAs）被认为在多种生物学过程中发挥着重要作用，包括细胞凋亡，这是卵泡闭锁的一个重要过程。我们旨在研究 circRNAs 是否参与颗粒细胞凋亡和由此导致的卵泡闭锁。我们从健康窦前卵泡（HA）和闭锁窦前卵泡（AA）中分离的猪颗粒细胞中生成了环状 RNA 表达谱。鉴定出超过 9632 个 circRNAs，其中 62 个 circRNAs 表达差异（DE-circRNAs）。验证了 circRNA 的反向剪接、RNase R 抗性和稳定性，并预测了 miRNA 结合位点和相关靶基因。选择了两个具有低错误发现率（FDR）、高倍数变化、miRNA 结合位点和相关生物学功能的外显子 circRNA（circ_CBFA2T2 和 circ_KIF16B）进行进一步表征。qRT-PCR 和线性回归分析证实了靶向基因（circ_CBFA2T2 的潜在靶基因）和凋亡基因（circ_KIF16B 的潜在靶基因）的表达和相关性。AA 卵泡颗粒细胞中 的 mRNA 含量增加，这进一步通过 p53 及其下游靶蛋白 pleckstrin homology like domain family a member 3（PHLDA3）在 AA 卵泡中的强烈免疫染色得到证实，而在 HA 卵泡中的颗粒细胞中则几乎没有染色。因此，我们得出结论，异常表达的 circRNAs 可能在卵泡闭锁中发挥潜在作用。
