Cell-free mitochondrial DNA as a potential predictor for hypoxic stress

Hypoxia exerts a far-reaching influence on cellular metabolic activities, which in turn has profound implications on cell functions. Emerging evidence has demonstrated that cell-free mitochondrial DNA (cf-mtDNA) exhibits a prompt response to acute stress within a relatively short span. Nevertheless, to date, there is a lack of reports on how hypoxic stress affects cf-mtDNA. Hypoxic stress cell models and mouse models were established. Western blot was used to examine the expression levels of HIF-1α and HIF-2α protein, and droplet digital PCR (ddPCR) was used to determine the copy numbers of cf-mtDNA in cell supernatants or in mice sera. Exosomes were isolated from the cell supernatants, with their characteristics analyzed by electron microscopy, nanoparticle tracking analysis (NTA), and Western blot. ELISA method was used to analyze the cortisol levels in mice sera. Receiver Operating Characteristic (ROC) curve was established to interpretate the ability of cf-mtDNA evaluating hypoxic stress. The cf-mtDNA copy number alterations are associated with hypoxic stress. Cf-mtDNA levels were found to increase under hypoxic stress, which was primarily derived from exosomes. Cf-mtDNA and cortisol levels exhibited an ascending tendency in hypoxic mice sera, with the AUC values for cf-mtDNA being 0.8690 on Day 3 and 0.8976 on Day 7, indicating that cf-mtDNA could serve as a potential predictor for hypoxic stress. The copy numbers of cf-mtDNA were increased under hypoxic stress. The extracellular release of cf-mtDNA was mainly derived from exosomes. Cf-mtDNA was proposed as a potential diagnostic predictor for acute mild hypoxic stress.