Identification of potential biomarkers associated with mitochondrial oxidative stress in idiopathic pulmonary arterial hypertension via bioinformatic and experimental analysis

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and devastating disorder. Mitochondrial oxidative stress has been found to be involved in PAH development. This study aims to explore potential mitochondrial oxidative stress-related biomarkers in IPAH via integrated bioinformatics, validate these genes in MCT-induced rat PAH model and examine the functional roles of COX6B1 and HMGCL in mitochondrial oxidative stress in PDGF-induced pulmonary arterial smooth muscle cells (PASMCs). GSE15197 was downloaded from GEO database. Genes associated with mitochondria and oxidative stress were obtained from Molecular Signatures Database and MitoCarta3.0 databases. Weighted gene co-expression network analysis (WGCNA) was used. GO and KEGG enrichment analysis were utilized. Furthermore, machine learning algorithms (LASSO, SVM-RFE and RF) were constructed to screen hub genes. A nomogram model based on the hub genes were performed for evaluation the diagnostic value, and the expression of hub genes were validated by real-time quantitative PCR and Western blot in MCT-induced rat PAH model. Finally, COX6B1 and HMGCL specific siRNAs were transfected into PDGF-induced PASMCs, mitochondrial ROS (mtROS) level and MnSOD activity were examined. A total of 18 genes related to mitochondrial oxidative stress were identified in IPAH. Two hub genes (COX6B1 and HMGCL) were finally determined as potential biomarkers. Based on the 2 genes, a nomogram model was constructed. This model exhibited remarkable diagnostic efficiency values for IPAH. The expressions of COX6B1 and HMGCL were found to be upregulated in MCT-induced PAH model, in agreement with the findings of bioinformatics analysis. While, transfection with either COX6B1 siRNA or HMGCL siRNA suppressed the abnormal accumulation of mtROS and restored MnSOD activity in PDGF-induced PASMCs. Our results indicated that two mitochondrial oxidative stress-related genes (COX6B1 and HMGCL) were identified as diagnostic biomarkers and may serve as potential targets for IPAH development, although further research is necessary.