The Second Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang 421001, Hunan Province, PR China; Hengyang Nanhua-Xinghui Reproductive Health Hospital, Hengyang 421001, Hunan Province, PR China.
Hengyang Medical School, University of South China, Hengyang 421001, Hunan Province, PR China.
J Reprod Immunol. 2022 Sep;153:103681. doi: 10.1016/j.jri.2022.103681. Epub 2022 Jul 31.
Hypercholesterolemia is defined as a high risk factor for causing female infertility by changing the cholesterol level in granulosa cells to impair the microenvironment of oocyte development and maturation. High blood levels of oxidized low-density lipoprotein (ox-LDL) undergoes an increase of autophagic granulosa cell death. Unfortunately, this underlying molecular mechanism remains largely elusive. We aim to uncover the role of circ-ubiquitin specific peptidase 36 (USP36) in autophagic granulosa cell death.
Exposure of ox-LDL on the ovarian granulosa cell-like human granulosa (KGN) cells line was established for simulating the situation of hypercholesterolemia in vitro. Levels of circUSP36 and ULK1 were detected using real-time polymerase chain reaction (RT-PCR). Cell viability and apoptosis were assessed using (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively. Immunofluorescence staining of LC3 was performed to evaluate activity of autophagy. Western blot was employed to determine expression of apoptosis and autophagy-associated markers. RNA immunoprecipitation (RIP) and RNA pull-down assays were subjected to verify the circUSP36-PTBP1-NEDD4L regulatory axis.
Treatment of ox-LDL induced aberrantly up-regulated circUSP36. Knockdown of circUSP36 alleviated cell apoptosis and excessive autophagy of granulosa cells triggered by ox-LDL. Mechanistically, reinforced expression of circUSP36 guided and facilitated PTBP1 binding to the coding region (CDS) of NEDD4L, resulting in NEDD4L mRNA decay. ULK1 was regulated by the circUSP36-PTBP1-NEDD4L axis in granulosa cells, thereby contributing to autophagic granulosa cell death.
In summary, ox-LDL fostered autophagic granulosa cell death through circUSP36-mediated NEDD4L mRNA decay, thus elevating ULK1 expression.
高胆固醇血症通过改变颗粒细胞中的胆固醇水平，从而损害卵母细胞发育和成熟的微环境，被定义为导致女性不孕的高危因素。氧化型低密度脂蛋白（ox-LDL）的血液水平升高会导致自噬性颗粒细胞死亡增加。不幸的是，这种潜在的分子机制在很大程度上仍未被揭示。我们旨在揭示环状泛素特异性肽酶 36（USP36）在自噬性颗粒细胞死亡中的作用。
通过体外模拟高胆固醇血症，建立了氧化型低密度脂蛋白（ox-LDL）对卵巢颗粒细胞样人颗粒细胞（KGN）细胞系的暴露。采用实时聚合酶链反应（RT-PCR）检测 circUSP36 和 ULK1 的水平。采用（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐（MTT）测定法、流式细胞术和末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）染色分别评估细胞活力和细胞凋亡。通过免疫荧光染色检测 LC3 评估自噬活性。采用 Western blot 检测凋亡和自噬相关标志物的表达。采用 RNA 免疫沉淀（RIP）和 RNA 下拉实验验证 circUSP36-PTBP1-NEDD4L 调节轴。
ox-LDL 处理诱导 circUSP36 异常上调。circUSP36 敲低减轻了 ox-LDL 诱导的颗粒细胞凋亡和过度自噬。机制上，circUSP36 的过表达指导并促进了 PTBP1 与 NEDD4L 的编码区（CDS）结合，导致 NEDD4L mRNA 降解。在颗粒细胞中，ULK1 受 circUSP36-PTBP1-NEDD4L 轴的调节，从而促进自噬性颗粒细胞死亡。
综上所述，ox-LDL 通过 circUSP36 介导的 NEDD4L mRNA 降解促进自噬性颗粒细胞死亡，从而提高 ULK1 的表达。
