Exploration potential regulatory mechanisms in ovarian injury of mice exposed to low-pressure hypoxia through whole transcriptome sequencing analysis [RNA-seq]

Chronic high-altitude hypoxia causes significant ovarian tissue damage. This study investigates the underlying mechanisms of ovarian injury in mice exposed to hypoxia, aiming to guide targeted therapy development. Mice were divided into either normal pressure (MP, n=15) or hypobaric hypoxia (PU, n=15) and samples from both groups were analyzed using whole-transcriptome sequencing data. Hub genes were identified through differential expression analysis and protein-protein interaction (PPI) network construction before their expression patterns were examined. Subsequently, functional enrichment was conducted. Key miRNAs, lncRNAs, circRNAs, and transcription factors (TFs) were ascertained. Finally, RT-qPCR and Western blotting (WB) were performed. Hmgcr, Ptgs2, and Mmp3 were identified as hub genes, all showing pronounced lower expression in PU samples. RT-qPCR analysis confirmed the downregulation of these genes, with WB analysis further demonstrating a significant reduction in their protein levels in PU samples. These hub genes were predominantly enriched in "oxidative phosphorylation" pathway. Furthermore, lncRNA-miRNA-mRNA, circRNA-miRNA-mRNA, and TF-mRNA regulatory networks were constructed. These networks highlighted key regulatory molecules, including the miRNA mmu-miR-144-3p, 2 lncRNAs (Miat and Neat1), 8 circRNAs (eg. novel_circ_041272-mu-miR-144-3p-Ptgs2), and 1 TF (Etv4). These results provide key insights for targeted ovarian injury therapies under low-pressure hypoxia. Overall design: Female 8-week-old C57BL/6 mice were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Animal License No.: SCXK (Beijing) 2024-0001). Notably, the mice were maintained under standard laboratory environment with a 12-hour light/dark cycle (07:00–19:00), ambient temperature maintained at 22±2°C, and a relative humidity of 45%–55%. The mice were provided with ad libitum access to standard laboratory chow and drinking water. The experimental animals were randomly divided into two groups: the MP group (Normal Pressure, n=15) and the PU group (Pressure Under Vacuum, n=15). The PU group was placed in a hypobaric hypoxia animal chamber (Guizhou Fenglei Aviation Mechanical Manufacturing Company, Guizhou, China) simulating the conditions of an altitude of 6500 meters, with a pressure of 43.12 kPa. The control group was housed in a normal environment at an altitude of 1588 meters and a pressure of 83.56 kPa, in Lanzhou, China. All mice were maintained for 15 consecutive days. All animal experiments were strictly conducted in compliance with the international ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments) and were approved by the Institutional Ethics Committee of The 940th Hospital of Joint Logistic Support Force of Chinese People's Liberation Army (Approval No. 2023KYLL299).Animal welfare measures were implemented according to the 2020 American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals.The detailed procedures were as follows: mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (40 mg/kg), followed by cervical dislocation performed by trained personnel, with subsequent collection of blood and ovarian tissue samples for analysis.These samples were used for evaluating the mouse model conditions and performing whole-transcriptome sequencing.