Re-analyzing and integration of omics-based expression profiles could be an advantageous tactic to catch a holistic view of the involving genes and miRNAs in the IgAN pathogenesis. Until now, several re-analyzes have been performed on the IgAN related expression profiles, mostly on kidney tissue samples of the patients [25-28]. As far as we know, there are just one experiment conducting a meta-analysis study seeking for DEGs in the PBMCs of IgAN patients. However, the mentioned experiment is suffering from lack of dataset quality control checking and not considering other regulatory elements like miRNAs, or lncRNAs in introducing hub-DEGs [29]. In the present study, following quality control checking, we performed re-analysis and integration of two expression datasets (mRNA and miRNA) related to PBMCs of IgAN patients. With applying a systems biology approach, we identified several key genes, TFs, miRNAs and their related lncRNAs which may have a potential role in the pathogenesis of IgAN. According to the results of pathway enrichment analysis, the intersecting mRNAs were mainly enriched in apoptosis, innate immunity and NGF signaling pathways.
P53 and XIAP (X-linked inhibitor of apoptosis protein), were among the top DEGs with the highest degree centralities in the regulatory network. These two proteins are considered as main participants in controlling the apoptosis and proliferation of cells. However, the activity of these two regulatory constituents are unlike; While p53 activity is in the favor of apoptosis and preventing the cell proliferation, XIAP action is to inhibit caspase 3, 7, and 9 and stop the apoptosis [30-32]. Considering the well-known roles of p53 and XIAP in controlling the apoptosis [33], these proteins might take a part in controlling the PBMCs proliferation in IgAN patients. In addition, similar to the previous results [34], our findings also pointed to the apoptotic phenotype of PBMCs in IgAN patients. Moreover, based on some evidence, P53 can suppress autoimmunity (e.g. systemic lupus erythematosus) by suppressing T cells activity while the precise mechanisms are still unknown [35].
Other than apoptosis, “innate immunity”, “toll like receptor cascades” and “interferon signaling” were other enriched pathways for the identified DEGs in the PBMCs of IgAN patients. Such enrichments were expected due to autoimmune nature of IgAN disease. Similar to previous findings, involvement of these pathways could be an indication for hyper-activation and enhancement of antigen processing pathways in PBMCs of IgAN patients [36, 37]. Dysregulation of innate immunity has also been comprehensively suggested in IgAN patients [38].
Nerve growth factor (NGF) signaling was another main enriched pathway for the DEGs in PBMCs of IgAN patients. NGF as a prototypical example of neurotrophic factors, is mainly involve in growth, proliferation and differentiation of neurons [39]. However, role of NGF in the proliferation of immune cells, stimulation of IgM, IgA and IgG production in lymphocytes, as well as regulation of inflammatory mediators like IL-1 beta, TNF-alpha , and IL-6 have been shown previously [40, 41]. Moreover, some findings have revealed mediatory role of NGF in autoimmune diseases like multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus [39]. Other findings also disclosed an increased serum levels of NGF, as well as higher NGF receptor expression in PBMCs of glomerulonephritis patients [42]. Findings of the present study also could be an evidence for the contribution of NGF signaling in the pathological process of IgAN disease.
Generally, involvement of all the above-mentioned pathways along with other enriched pathways like notch signaling and EGFR pathways could point to an aberrant proliferation rate and distortion of PBMCs in the IgAN patients. Involvement of the enriched pathways may provide new explanations about the underlying immunopathological mechanisms of IgAN.
In the next step, a multilayer regulatory network was constructed to identify different hub molecules as the main potential participants in the pathogenesis of the disease. The concept of construction a multilayer network was to achieve a holistic aspect of regulatory interactions in the PBMCs of IgAN patients. Similar experiments in this area of research only consider DEGs to construct an interactive regulatory network, while skipping other regulatory factors like TFs, miRNAs and lncRNAs. Such studies suffer from a narrow view and ignore the complex regulatory nature of the cell. Accordingly, a multilayer regulatory network is thought to be more realistic and closer to the complex nature of cellular regulatory machineries. The key regulatory constituents in the multilayer network are listed in table 2. Here, we introduced top DEGs with the highest degree scores in the network as potential biomarker/therapeutic targets. Moreover, in case of TFs, Nuclear actor Kappa B Subunit 1 (NF-κB) with a highest degree among other TFs, identified as one of the key TFs in regulating the expression of DEGs in IgAN patients. The trace of NF-κB in IgAN pathogenicity has been previously shown, where Toll-like receptors trigger a cascade of intracellular massages that finally leads to the activation of this transcription regulator [43]. The consequence of such activation is cellular release of different cytokines and chemokines, which may participate in aberrant galactosylation of IgA1 [44].
MiRNAs as other main regulatory elements also could play a major role in immunological and pathological aspects of IgAN. Along with their regulatory roles, a higher stability in biological milieu makes these small RNAs as potent non-invasive biomarkers for early detection of diseases. In case of DEmiRs in the constructed regulatory network, most of the top miRNAs including miR-16, miR-92, miR-17, and let-7b have shown to play a direct role in controlling the cell cycle and cell proliferation [45-48]. miR-124-3p as another hub-DEmiR in the regulatory network was also among the top 10 DEmiRs in the DEmiR dataset (log2FC: 4.7). According to previous experiments, miR-124 has a crucial role in innate immunity system and fine tuning the toll-like receptor responses via targeting signal transducer and activator of transcription 3 (STAT3) [43]. The results of our analysis also pointed to the negative regulatory role of miR-124 in inflammatory responses and introduced this miRNA as a therapeutic target, as well as a non-invasive biomarker for detection of IgAN disease.
Back to the multilayer regulatory network and talking about regulatory roles of miRNAs, let-7b was introduced as another top DEmiR able to target a number of DEGs and thought to play a pivotal role in IgAN process/pathogenesis. Up-regulation of this miRNA in PBMCs of IgAN patients and its role in the glycosylation process of IgA with regulation of N-acetylgalactosaminyltransferase 2 (GALNT2) enzyme was previously shown [49, 50]. Overexpression of let-7b in PBMCs of IgAN patients might be a reason for the reduced expression levels of GALNT2 enzyme (log2FC: -4.43 in the DEG dataset), and consequently aberrant galactosylation of IgA1 in patients. As a result, based on this analysis and previous findings, this miRNA could be considered as a potential therapeutic target for IgAN.
Another layer of the constructed network was specified to lncRNAs as another regulatory molecules showing an outstanding potential in pre- and post-transcriptional regulations [24]. Despite the regulatory role of lncRNAs, their biomarker potential in different types of kidney diseases like membrane nephropathy [51], diabetic nephropathy [52] and IgAN nephropathy [53] have been proposed by several research teams. In this investigation, after importing different curated lncRNAs in the constructed network, HOTAIR was introduced as the topmost lncRNA in regulating the DEGs and DEmiRs in IgAN pathogenicity.
HOTAIR as an anti-sense lncRNA is involved in epigenetic silencing of various genes by recruiting the PRC2 complex, trimethylation of H3K27 across the HOXD locus and finally repressing the transcription [54]. According to recent findings, transcriptional repression of this RNA molecules in immune cells could lead to NF‐κB activation and consequently inflammatory responses [55]. Likewise, the potential role of HOTAIR in provoking inflammation was observed in a study on rheumatoid arthritis patients [56]. Here, we also proposed the probable role of this lncRNA in IgAN disease and introduced this RNA molecule as a key target of further analysis in IgAN disease.