Identification and Verification of Necroptosis-Related Genes in Patients With Sepsis by Bioinformatic Analysis and Molecular Experiments

Although necroptosis is an emerging mechanism of multiple organ dysfunction in sepsis, data on the mechanistic link between necroptosis and sepsis are scarce. Bioinformatic analysis was performed to compare the gene profiles between the sepsis (n = 133) and healthy control (n = 12) groups and identify necroptosis-related differentially expressed genes (DEGs). The identified necroptosis-related DEGs were verified by three-step molecular experiments: (1) quantitative real-time PCR and enzyme-linked immunosorbent assay; (2) cell culture, transfection and Western blotting; and (3) cytokine array with apoptosis inhibition. Additionally, receiver-operating characteristic curve analyses were performed to evaluate the performance of the corresponding proteins to the necroptosis-related DEGs in diagnosing sepsis and in predicting in-hospital mortality of patients with sepsis. Eight necroptosis-related DEGs, including five upregulated (PYGL, TNF, CYLD, FADD and TLR3) and three downregulated (TP53, FASLG and NLRP6) DEGs, were identified. Moreover, the levels of the corresponding proteins to necroptosis-related DEGs showed excellent or considerable accuracy in diagnosing sepsis and in predicting the mortality of sepsis patients. In cell culture media transfected with plasma from the sepsis and control groups, Western blotting revealed that the levels of the corresponding proteins were increased in the upregulated DEGs and decreased in the downregulated DEGs. The cytokine array revealed cytokines in cell culture media transfected with plasma from patients with sepsis while preventing apoptosis by inhibiting the caspase-8 activity, wherein the transfected cells potentially underwent necroptosis. Eight necroptosis-related DEGs were identified in patients with sepsis by bioinformatic analysis and verified by molecular experiments, implying that necroptosis may be a key mechanism of sepsis.