Reproductive Medical Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China.
Department of Human Anatomy, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China.
J Cell Physiol. 2021 Jul;236(7):5162-5175. doi: 10.1002/jcp.30222. Epub 2021 Jan 3.
Long noncoding RNA PVT1 is involved in the progression of female gynecological cancers. However, the role of PVT1 in ovarian granulosa cell apoptosis-mediated premature ovarian insufficiency (POI) remains unclear. This study aims to elucidate the role of PVT1 in ovarian granulosa cell apoptosis-mediated POI. The expression of PVT1 was compared between ovarian tissues from POI patients and normal controls. The methylation level in the PVT1 promoter region was detected by methylation-specific polymerase chain reaction. The interaction between PVT1 and forkhead box class O3A (Foxo3a) was confirmed by RNA pull-down and RNA immunoprecipitation assays. Granulosa cell apoptosis was detected using flow cytometry. The effect of PVT1 on transcription activity of Foxo3a was detected by luciferase reporter assay. The expression of PVT1 was low in the POI ovarian tissues compared with the controls, and such a low expression was related to the hypermethylation of the PVT1 promoter. PVT1 was localized in both the cytoplasm and the nucleus of granulosa cells. We determined that PVT1 could bind with Foxo3a and that downregulating PVT1 by small interfering RNAs inhibited Foxo3a phosphorylation by promoting SCP4-mediated Foxo3a dephosphorylation, resulting in an increase in Foxo3a transcription activity. Moreover, downregulating PVT1 promoted granulosa cell apoptosis by increasing the Foxo3a protein levels. An in vivo experiment showed that the injection of PVT1 overexpressing vectors restored the ovarian function in POI mice. Hypermethylation-induced downregulation of PVT1 promotes granulosa cell apoptosis in POI by inhibiting Foxo3a phosphorylation and increases the Foxo3a transcription activity.
长链非编码 RNA PVT1 参与妇科癌症的进展。然而，PVT1 在卵巢颗粒细胞凋亡介导的卵巢早衰（POI）中的作用尚不清楚。本研究旨在阐明 PVT1 在卵巢颗粒细胞凋亡介导的 POI 中的作用。比较 POI 患者和正常对照的卵巢组织中 PVT1 的表达。通过甲基化特异性聚合酶链反应检测 PVT1 启动子区域的甲基化水平。通过 RNA 下拉和 RNA 免疫沉淀实验证实 PVT1 与叉头框 O3A（Foxo3a）的相互作用。通过流式细胞术检测颗粒细胞凋亡。通过荧光素酶报告基因检测检测 PVT1 对 Foxo3a 转录活性的影响。与对照组相比，POI 卵巢组织中 PVT1 的表达较低，这种低表达与 PVT1 启动子的高甲基化有关。PVT1 定位于颗粒细胞的细胞质和细胞核中。我们确定 PVT1 可以与 Foxo3a 结合，通过小干扰 RNA 下调 PVT1 可通过促进 SCP4 介导的 Foxo3a 去磷酸化来抑制 Foxo3a 磷酸化，从而增加 Foxo3a 转录活性。此外，下调 PVT1 通过增加 Foxo3a 蛋白水平促进颗粒细胞凋亡。体内实验表明，过表达 PVT1 载体的注射恢复了 POI 小鼠的卵巢功能。高甲基化诱导的 PVT1 下调通过抑制 Foxo3a 磷酸化促进 POI 中的颗粒细胞凋亡，并增加 Foxo3a 转录活性。
