POAG is a chronic retinal neurodegeneration disease characterized by changes in the anterior and posterior segments of the eye; in addition, serious damage may be detected in the TM and ONH [1, 5, 8]. Lowering IOP using drugs or surgery is the only intervention currently available . However, clinical evidence indicates that lowering IOP does not prevent progression in all POAG patients. Consequently, non-IOP factors are involved in the disease . With the rapid development of science and technology, bioinformatics provides a powerful strategy for the screening of molecular markers . Biomarkers reflect changes at the molecular level and can accurately monitor pathological changes in the TM and ONH and provide important information for the diagnosis of POAG [5, 15, 19,20,21,22,23]. Nevertheless, the detection of POAG lesions using molecular biology methods is suboptimal, and treatments are currently in a limited pharmacotherapy phase .
In the present study, to screen the pathogenic genes involved in POAG, an integrated analysis was performed by using microarray datasets in glaucoma derived from the GEO database. The functional annotation and potential pathways of DEGs were additionally examined by GO and KEGG enrichment analyses. A POAG-specific transcriptional regulatory network was constructed to identify crucial transcription factors that target the key genes in patients with POAG. We used random forest and the SVM method to screen five potential key genes in both human TM and ONH tissue. A growing number of researchers realize that immune infiltration is related to the diagnosis of POAG [10,11,12]. Therefore, analyzing the pattern of POAG immune cell infiltration and finding specific diagnostic markers have profound significance for POAG patients. Subsequently, the CIBERSORT algorithm performed deconvolution analysis on the immune microenvironment to assess the proportion of immune cells in POAG.
We identified key differentially expressed genes between POAG and normal tissues by performing a combined analysis of TM and ONH. A total of 49 DEGs were identified, including 19 downregulated genes and 30 upregulated genes. These 49 differentially expressed genes were further analyzed by random forest and SVM to screen the key genes. Five genes (KRT14, HBB, ACOX2, HEPH and KRT13) were significantly changed. We found that these key genes were highly correlated with immune cells. The immune microenvironment is composed of a variety of lymphocytes, such as T cells, B cells and macrophages, etc. . From the immune infiltration analysis, we found that there was a significant difference in the relative cell content of 22 types of immune cells (e.g., B cells naïve, B cell memory, plasma cells, T cells CD8, T cells CD4 naïve, T cells CD4 memory resting) in normal control samples versus in POAG samples.
KRT14 is a member of the type I keratin family of intermediate filament proteins . KRT 14 is expressed in a variety of cells in humans. It is recovered as a heterodimer with KRT5 and forms the cytoskeleton of epithelial cells [26, 27]. Gautam et al., using multispecies single-cell transcriptomic analysis of the human eye, found that KRT 14 was expressed in corneal epithelial cells . However, previous studies did not perform a deeper investigation of POAG. In our study, we found that KRT14 was expressed in both TM and ON tissues in humans. KRT14 was highly correlated with immune cells (plasma cells, neutrophils, etc., and negatively correlated with regulatory T cells (Tregs) and mast cell resetting. HBB (hemoglobin subunit beta) is encoded by the HBB gene on human chromosome 11 . HBB was positively correlated with activated NK cells and monocytes and negatively correlated with resting mast cells and resting dendritic cells. The ACOX2 gene encodes the enzyme Acyl-CoA oxidase 2 in human autosome 3, oxidizing the Coenzyme A esters of bile acid and di-trihydroxycholestanoic acid intermediates. It is located in the peroxisomes of cells and has a tripeptide at the C-terminal end of the protein formed by serine-lysine-leucine that serves as a peroxisome localization signal. Deficiency of this enzyme causes an accumulation of fatty acids and bile intermediates, generating Zellweger syndrome. ACOX2 was positively correlated with CD4 memory resting T cells and monocytes and negatively correlated with cellular helper T cells and naïve CD4 T cells. HEPH (Hephaestin) is involved in the metabolism and homeostasis of iron and possibly copper . It is a transmembrane copper-dependent ferroxidase responsible for transporting dietary iron from intestinal enterocytes into the circulatory system. HEPH was positively correlated with memory CD4 + T-cell resetting and regulatory T cells (Tregs) and negatively correlated with naive CD4 + T cells and follicular helper T cells. The KRT13 (keratin 13) gene encodes a type I cytokeratin that is expressed in the differentiated cells of noncornified stratified squamous epithelia [30, 31]. KRT13 is positively correlated with T-cell follicular helper and plasma cells and negatively correlated with T-cell regulatory (Tregs) and resting mast cells.
The ocular innate immune response relevant to glaucoma involves the complement cascade, microglia, astrocytes, and Müller cells. Autoantibodies, tumor necrosis factor (TNF)-alpha, and other products of adaptive B and T cells are also featured prominently in the glaucomatous adaptive immune response. However, research has not provided clear immune infiltration among these cells . Thus, in our study, based on the immune infiltration analysis between key genes and immune cells, we divided the differentially expressed mRNAs into upregulated and downregulated groups and predicted the drug targets of the differentially expressed genes through the Connectivity Map database. The results showed that the expression profiles of drug disturbances such as avrainvillamide-analysis-3, cytochalasin-D, NPI-2358, oxymethylone and vinorelbine were negatively correlated with the expression profiles of disease disturbances. NPI-2358 and vinorelbine are tubulin inhibitors. Cytochalasin-d is an actin polymerization inhibitor. Oxymetholone is an androgen receptor agonist. avrainvillamide-analog-3is a nucleophosmin inhibitor. This finding indicated that these drugs may reduce or even reverse the POAG disease state.
There were several limitations to this study. First, due to the limited number of samples, there is still a need to confirm these preclinical observations in future clinical studies for novel biomarkers. Second, CIBERSORT is based on the principle of linear support vector regression and uses gene expression data in reverse to deduce the result of immune cell infiltration. Indeed, it is not based on experimental data, and further verification of immune cell infiltration by a large number of experiments is needed. Third, we performed mining and analysis of previously published data; although some previous studies showed similar results, the related molecules and their mechanisms at the molecular, cell, and tissue levels require validation.
In conclusion, the overlap of the random forest and SVM algorithms was obtained, and five key genes were eventually recognized in both human TM and ONH tissue. We found that it may be used as a diagnostic marker for POAG. To understand POAG development, GO and GSEA of the selected genes supplied a more specific molecular mechanism. To date, the relationship between key genes and immune infiltration in both TM and ONH tissues has been rarely reported. The mechanism of key genes and immune infiltration-related factors in the diagnosis of POAG remains to be explored. Further investigation of these immune cells may identify targets of immunotherapy for POAG and help POAG patients benefit from immunomodulatory therapy.