In this study, an insight of gene expressions and their related proteins interactions in childhood allergic asthma was discovered based on the combination of genome array study and bioinformatics analysis. A total of 269 DEGs have been identified and 200 of them are up-regulated genes and the rest of them are down regulated genes. After functional analysis and PPI network construction, several pathways and hub genes related to childhood asthma were identified.
In the top 10 up-regulated and down-regulated DEGs and hub genes, we found several DEG that may be related to childhood allergic asthma.
Small nucleolar RNA, C/D box 46 (SNORD46) was one of the top 10 up-regulated DEGs in childhood allergic asthma with logFC = 2.04. According to a recent study, SNORD46 functions as an oncogene in the lung cancer [19]. Other experiments identified that SNORD46 overexpressed in several types of cancer including lung cancer and regulated the cell proliferation, viability, impaired cell migration and invasion in lung cancer [20]. Airway narrowing is considered as the major symptom of asthma, which is caused by airway smooth muscle hyperplasia and hypertrophy (especially hyperplasia) [21]. SNORD46 was suspected of involvement in the airway smooth muscle hyperplasia of asthma. However, there are no researches focusing on SNORD46 and asthma. Further studies are needed to explore whether SNORD46 participates in the airway smooth muscle hyperplasia of asthma.
Ribosomal Protein S3 (RPS3) gene was not only top 10 up-regulated DEGs (logFC = 1.64), but also one of the top 10 hub genes (score = 20). RPS3 is a ribosomal protein that plays a crucial role in the NF-κB pathway, which binds to the p65 subunit so the NF-κB complex’s nuclear translocation will be promoted and the binding ability of p65 will be strengthened [22]. NF-κB pathway mainly regulates the inflammation response and cellular activities like cell proliferation and cell death [23]. According to a study in 2012, the RPS3-p65 interaction in NF-κB pathway is missing due to the knockout of RPS3 and it could significantly decrease the cytokine production in B cells, T cells proliferation and immunoglobulin κ light chain gene expression in B cells [24]. Also another research has confirmed that RPS3-p65 interaction influenced a lot in the immune response by regulating cytokine production and proliferation in T cells and immunoglobulin κ light chain gene expression in B cells [25]. High grade of immune responses such as high level of IgE, hypersecretion of mucus and high level of Th2 cytokines are characteristics of allergic asthma [26]. We hypothesized that the overexpression of RPS3 in NF-κB pathway elevates immune response in individuals and results in allergic asthma. Moreover, it was found in a mouse study that the lung RPS3 protein level was significantly increased in mouse asthma and RPS3 silence RNA remarkably inhibited allergic airway inflammation [27], and this study provided evidence for our hypothesis but further research detecting relationship between human allergic asthma and RPS3 is required.
There are three down-regulated genes: OR5E1P (logFC=-1.14), OR56A5 (logFC=-1.12) and OR51B6 (logFC=-1.08) coming from the olfactory receptor family, but their subfamilies are different. There are no current studies showing that these three genes are associated with asthma. However, a study in 2019 indicates that OR2AG2 from olfactory receptor family participates in the asthma pathogenesis and the suppression or genetic defects in OR2AG2 contribute to asthma pathogenesis [28]. According to the study, olfactory receptors express in the airways which is similar to bitter taste receptor [28]. Besides, another study shows that OR2AG1 (paralog of OR2AG2) and OR1D2 are functionally expressed in the in human airway smooth muscle cells (HASMCs) [29]. These two olfactory receptor genes are activated by different ligands [30, 31], which induce the brief increase of Ca2 + in HASMCs and both receptors can touch off cAMP-dependent signal transduction cascade [28]. CAMP possesses the ability to antagonize HASMC contraction and inhibits the cell proliferation, fights the airway narrowing [32]. In this way, cAMP can combat against asthma, and the activation of OR2AG1 and OR1D2 can trigger the inhibition of cell contraction by activating cAMP-dependent signaling pathway. Taken together, the deficiency or down-regulated of olfactory receptors: OR2AG2, OR2AG1 and OR1D2 play roles in asthma pathogenesis. Based on this, we hypothesize that the three down-regulated olfactory receptors in our study (OR5E1P, OR56A5 and OR51B6) have the similar characteristics like olfactory receptors (OR2AG2, OR2AG1 and OR1D2) and take part in the childhood allergic asthma pathogenesis. Since there are no current studies indicating that there exist relationships between these genes and asthma, further study is needed to confirm our hypothesis and investigate their effects in childhood allergic asthma pathogenesis.
MKI67 is a down-regulated gene (logFC=-0.55) and one of the top 10 hub genes. It is also known as the Ki-67, a nuclear DNA binding protein that used as a marker for tumor grading [33]. Ki-67 is a marker of cellular cycle and proliferation [34], and it can be detected in G1, S, G2 and M phase of cell cycle [35]. Ki-67 is regulated by phosphorylation and only non-phosphorylation Ki-67 can form complex with DNA and functions in proliferation cells [36]. Besides this, Ki-67 can interact with RNA-binding protein during the mitosis [37]. According to one published study, CDK-1 phosphorylates Ki-67 during mitosis [38], and the transition from G1 to S is mediated by Ki-67. Taken together, Ki-67 plays an essential role in the cell cycle which regulates the cell proliferation. Since proliferation may cause the hyperplasia of airway which results in major symptom of asthma, we suspect that Ki-67 may participate in asthma pathogenesis. However, in our study, we identified Ki-67 as a down-regulated DEG in the childhood allergic asthma patients. Another research in 2010 detects 2 biomarkers including Ki-67 between severe asthma patients and healthy controls, and it is found that the proliferating cell nuclear antigen and Ki-67 were highly expressed [39]. The disagreement might be induced by different ages of patients or different subtypes of asthma. Further experiment is needed to clarify our results.
Besides two genes mentioned above (MKI67 and RPS3), PDCD11 up-regulated gene (logFC = 0.73) is also one of the top 10 hub genes (score = 18). According to studies, PDCD11 induces the activation of NF-κB pathway and results the apoptosis of cells [40]. PDCD11 inducing the apoptosis by activating the transcription of the promoter [41]. Since NF-κB pathway mainly regulates the inflammation response and cellular activities like cell proliferation and cell death [41], the up-regulated gene PDCD11 may enhance the function of NF-κB pathway. We suspected that the overexpression of PDCD11 activates the NF-κB pathway, and leads to high inflammation response and cellular activities. This may contribute to the pathogenesis of allergic asthma. However, there is no study indicating the relationship between PDCD11 and childhood allergic asthma. Further research is required to detect the role of PDCD11 in childhood allergic asthma.
In addition to genes including up or down regulating genes and hub genes, pathways were also identified and discussed.
According to the GO and KEGG enrichment analysis, only one KEGG pathway enriched in childhood allergic asthma: Olfactory transduction. Olfactory receptors play a critical role in the olfactory transduction and also have the ability to regulate important physiological effects and control the intracellular Ca2 + level [29]. Ca2 + level are critical for the airway smooth muscle cell contraction which is related to allergic asthma, since the HASMCs of asthmatics often show hypercontractility [42]. Olfactory receptor expresses differently in the human airway smooth muscle cells [43] and regulates the Ca2 + level, so we suspected that the pathway of olfactory transduction and its regulated DEGs (OR5E1P, OR56A5 and OR51B6 etc.) may participate in childhood allergic asthma.
In GO enrichment analysis, cellular response to hydrogen peroxide (H2O2) is an enriched biological process. A significant characteristic of airway inflammation in asthma patients is the activation of multiple inflammatory cells including eosinophils [44]. Airway inflammation of asthma is correlated to oxidative stress, which exists in the high oxidant activity [45]. H2O2 is a mediator of oxidative stress released by eosinophils to enlarge the process of airway inflammation [46]. Airway smooth muscle stimulated by H2O2 and contract, narrowing the airway which induces asthma [47]. According to study, H2O2 level is significantly higher in the asthma patients than normal controls [48]. Taken together, we concluded that cellular response is an important biological process in childhood patients. Since the cells like airway smooth muscle cells responding to H2O2 can induce the contraction which may narrow the airway and lead to asthma.
However, limitations are still occur in the study and need to be recognized. First, only one dataset from one database (GEO) was used during the study which reduces the credibility of the results. Second, the information of the samples including ages, regions and races of the patients remained unknown. Moreover, according to the results, there are 10 hub genes but not all of them have clear regulated mechanisms in asthma. Therefore, more evidence based on experiments are needed to investigate the mechanisms. Finally, we did not perform clinical researches to support our results.
In summary, we have discussed genes from CD4 + T cells: SNORD46 (top 10 up-regulated genes), RPS3 (both top 10 up-regulated genes and top 10 hub genes), OR5E1P, OR56A5 and OR51B6 (three down-regulated genes from same family), MKI67 (down-regulated hub gene) and PDCD11 (up-regulated hub gene). Besides, olfactory transduction pathway and biological process cellular response to H2O2 have been discussed. Their importance and potential participation in childhood allergic asthma have been raised. Despite some of them need further research to confirm their roles or importance in childhood allergic asthma, our study provides inspiration for other researchers on DEGs and pathways in CD4 + T cells of childhood allergic asthma.