In this study, a WGCNA on mRNA expression profile GSE117261 downloaded from the GEO database was performed. Based on the WGCNA analysis, all 2299 DEGs obtained by the “limma” package in R software were clustered into 7 modules. Then, 597 DEGs in the yellow module were found to be the most positive genes related to PAH (correlation score = 0.74, P < 0.0001), which were mainly enriched in ‘cytoplasm’ and closely related to ‘transcription, DNA-templated’, ‘protein binding’ and ‘pathways in cancer’. GSEA showed that the gene set ‘HALLMARK_MYC_TARGETS_V1’ was obviously enriched in the PAH group. Four hub genes, vascular endothelial growth factor A (VEGFA), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), PNN Interacting Serine and Arginine Rich Protein (PNISR) and Heterogeneous Nuclear Ribonucleoprotein H1 (HNRNPH1) were determined based on the PPI network. Meanwhile, upregulatd expressions of PNISR and HNRNPH1 were validated by RT-qPCR.
More and more studies showed that cell proliferation was one of the pathophysiological mechanisms of pulmonary arterial hypertension[20]. Inhibition of the proliferation of pulmonary artery smooth muscle cells can effectively ameliorate pulmonary arterial hypertension[21]. We are delighted to find that cell proliferation is enriched in PAH in accordance with the previous findings.
Based on the GO analysis, the DEGs mainly consist of “Transcription, DNA-templated” in BP terms, which has been previously reported to be correlated to PAH[22]. The CC term “cytoplasm” and the MF term “protein binding” were both closely related to the transcription, which is an essential part of gene expression[23]. KEGG analysis revealed that the “Pathway in cancer” played a marked role in PAH, which was consistent with the previous demonstration demonstrated that the pathobiology of small vessels in severe PAH patients was quasi-neoplastic and cancer-like[24]. Meanwhile, the reliability of the DEGs obtained from the previous analysis was completely confirmed by results of the enrichment analysis.
By performing the GSEA analysis on the gene profile of GSE, many gene sets highly enriched in the PAH were found. The top six gene sets, including ‘HALLMARK_MYC_TARGETS_V1’, ‘E2F_TARGETS’, ‘MTORC1_SIGNALING’, ‘DNA_REPAIR’, ‘G2M_CHECKPOINT’, and ‘GLYCOLYSIS’ pathway, are cell cycle-related pathways, suggesting that genes involved in these pathways might contribute to cell proliferation. Among them, ‘HALLMARK_MYC_TARGETS_V1’ with an enrichment score of 0.59, was experimentally validated. This gene set involved 184 genes, including the well-known proliferating cell nuclear antigen (PCNA), minichromosome maintenance protein (MCM) and other cell proliferation markers[25]. Moreover, v-myc avian myelocytomatosis viral oncogene homolog (MYC) is a transcription factor known to regulate various human genes, promoting cell growth and proliferation[26], regulating apoptosis by altering pro- and anti-apoptotic members of the BCL-2 family and activating telomerase, controling the angiogenesis by regulating the expression levels of VEGF[27].
Based on previous studies, our work provides a new insight into the underlying pathogenesis of PAH. Dong H et al. identified CSF3R, NT5E, ANGPT2, FGF7 and CXCL9 as candidate biomarkers of PAH, and ruxolitinib might exert promising therapeutic action for PAH[28]. Qiu X et al. performed a WGCNA on GSE15197 and found that 11 real hub genes, including EP300, MMP2, CDH2, CDK2, GNG10, ALB, SMC2, DHX15, CUL3, BTBD1, and LTN1, were over-expressed in IPAH[29]. Contrastly, Liu J et al. suggested that 10 hub genes were determined via Cytohubba and a crucially ceRNA network was identified, including 14 LncRNAs, 2 miRNAs, and 3 mRNAs[30]. Interestingly, Li Q et al. confirmed that 9 hub genes related to PAH, particularly the PLK4 and SMC2 genes, providing a deeper understanding of physiopathologic of PAH [22]. Farha S et al. inferred that inhibition of KIT progenitor could improve remodeling and proliferation in PAH. Imatinib, a tyrosine kinase inhibitor that targets c-KIT, has been shown to be beneficial to PAH patients due to its inhibition of proliferation in hematopoietic progenitors and mast cells [31]. Liu J et al. found that VEGF initiated vascular remodeling resulting in PAH[32]. However, to our knowledge, research on the role of PNISR and HNRNPH1 in PAH remains limited.
PNISR is a newly identified serine-arginine (SR) protein in human that co-purifies with pinin, which is rarely reported[33]. Sinclair PB et al. suggested that abnormal expressions of GRIK2 and PNISR were associated with proliferation in some lymphoid leukemias[34]. HNRNPH1, a core member of the heterogeneous nuclear ribonucleo-proteins family, frequently upregulated in various cancer cells and contributed to tumorigenesis[35]. Elevated expression of HNRNPH1 was found in acute myeloid leukemia (AML), and knockdown of HNRNPH1 alleviated cell proliferation[36]. And the upregulated expressions of PNISR and HNRNPH1 in PAH were found in our study. According to the ROC curve, the values of the AUC of PNISR and HNRNPH1 were 0.815 and 0.744, respectively. The AUC result indicated that PNISR and HNRNPH1 had a powerful ability to discriminate PAH from the controls. Together, PNISR and HNRNPH1 were considerated as candidate biomarkers of PAH. Nevertheless, further researches are required to investigate the role of PNISR and HNRNPH1 on the development of PAH, and the expansion of the sample size is needed to validate the efficacy of PNISR and HNRNPH1 as biomarkers for PAH.