Study on potential differentially expressed genes in stroke by bioinformatics analysis

The incidence of IS is caused by a combination of many factors, such as environment and heredity. In this study, the gene chip information of peripheral blood mononuclear cells of IS patients and healthy controls was obtained by using GEO database, and 85 DEGs were analyzed and screened by using GEO2R software, including 65 upregulated genes and 20 downregulated genes. Then, GO function enrichment analysis and KEGG pathway enrichment analysis were carried out on the DEGs obtained. The results of GO functional classification showed that DEGs mainly concentrates on signal transduction, immune response, inflammatory response, and receptor binding. In the PPI network of DEGs, the scores of CEACAM8, CD19, and MMP9 were higher. The results of KEGG pathway enrichment analysis showed that DEGs mainly mediated cytokine-receptor interaction, hematopoietic pathway, and B cell receptor signaling pathway.

Adhesion molecule is a kind of membrane surface glycoprotein which can mediate the adhesion between cell–cell and cell-extracellular matrix. It is mainly expressed in leukocytes, platelets, and endothelial cells. There are many kinds of adhesion molecules including platelet membrane glycoprotein [11]. At present, the view that inflammation and immune response play a key role in cerebrovascular diseases has been widely recognized. Inflammatory cells can lead to the formation of early pathological changes of cerebrovascular diseases, inflammatory effector molecules can lead to the progression of pathological changes, and inflammatory activation can lead to the occurrence of acute ischemic cerebrovascular diseases. The adhesion of circulating leukocytes to endothelial cells and migration into arterial walls is an early step in the formation of atherosclerosis, which needs to be mediated by cell adhesion molecules expressed on the surface of vascular endothelial cells. Adhesion molecules allow monocytes and lymphocytes to roll, adhere tightly, and migrate across endothelium. Adhesion molecules play an important role in the occurrence of stroke and cerebral ischemia–reperfusion (I/R) injury. The adhesion in vascular endothelial cells and transmembrane migration of leukocytes need to be mediated by adhesion molecules, so adhesion molecules are an important inflammatory mediator. In addition, platelet activation is also related to adhesion molecules. At present, it is considered that cerebral I/R injury is essentially an inflammatory process, in which white blood cells infiltrate into brain tissue through the interaction with adhesion molecules. Therefore, the treatment measures related to anti-adhesion molecules were expected to become a new field in the treatment of cerebrovascular diseases [12, 13]. In addition, adhesion molecules can reflect whether vascular endothelial cells are damaged or not, and can also reflect the activation state of leukocytes and platelets, which all play a role in the occurrence and development of cerebrovascular diseases. Therefore, adhesion molecules can be used as predictors of stroke in theory. The correlation between adhesion molecules and cerebrovascular diseases has been preliminarily confirmed, and the determination of adhesion molecules may have potential value in preventing and monitoring cardiovascular and cerebrovascular diseases, guiding clinical treatment.

CD19 is a member of B-cell-specific immunoglobulin superfamily, which is expressed by early B cells during the period from heavy chain rearrangement to plasma cell differentiation. CD19 has been reported to enhance the activity of Src family protein tyrosine kinase and mitogen-activated protein kinase, promote cell proliferation, and positively regulate the function of B cells. Its functional diversity and important signal transduction make it play an important role. If abnormal expression occurs, it will lead to B cell–related diseases [14]. In addition, it can mediate the pathophysiological process of diseases by affecting immune function.

Matrix metalloproteinase families (MMPs) are closely related to cerebrovascular diseases. Some studies have shown that [15] there is a certain relationship between acute ischemic stroke and MMPs. By detecting patients with acute ischemic stroke, it is found that the level of MMPs in serum will obviously increase. Elevated MMPs can degrade extracellular matrix, aggravate vascular brain edema, and damage neurons. The experiment found that [16] knocking out MMPs gene or adding MMPs inhibitor can obviously reduce the injury of stroke. MMP9 is one of the most closely related members of MMPs family with cerebrovascular diseases, which can degrade the components of cerebrovascular basement membrane, increase the permeability of blood–brain barrier, and aggravate the occurrence of cerebral edema after being activated. The research also confirmed that the high expression of MMP9 can aggravate the destruction of blood–brain barrier and promote the occurrence of brain edema, and the purpose of relieving brain edema can be achieved by knocking out MMP9 in mice or using MMPs inhibitors [17]. In the central nervous system diseases such as inflammation, MMP9 could lead to cerebral hemorrhage and brain edema by breaking the blood–brain barrier, and had toxic and side effects on neurons in varying degrees [18]. The results of clinical research on patients with cerebral ischemia suggested that the expression level of MMP9 in human cerebral ischemia patients was higher than that in healthy people [19], and the expression level of MMP9 was also closely related to the prognosis of patients with cerebral ischemia. There is a certain relationship between the expression level of MMP9 and the hemorrhagic transformation after cerebral ischemia, the expression level of MMP9 can predict the possibility of transforming into cerebral hemorrhage. After later observation, it was found that the plasma level of MMP9 was higher in patients with hemorrhagic transformation after cerebral hemorrhage. After cerebral ischemia, the degranulation of neutrophils leads to the release of stored MMP9, which leads to the increase of blood MMP9 level. Therefore, MMP9 can be used as a blood detection marker in the prediction of hemorrhagic transformation of patients, and the high expression level of MMP9 after cerebral ischemia is more likely to lead to hemorrhagic transformation after ischemia [20]. The disorder of MMP9 gene level may participate in the pathophysiological process of stroke, and may become a new molecular target for diagnosis and prognosis of stroke patients.

The results of GO functional enrichment analysis showed that the biological processes involved in DEGs were mainly concentrated in signal transduction, immune response, inflammatory response, and cytokine-receptor interaction, suggesting that inflammatory response played an important role in the occurrence and development of IS. Pathological inflammation can cause permanent damage to functional cells and vegetative cells in the brain, and the damage caused by IS could be alleviated by inhibiting inflammation [21]. In the rat model of middle cerebral artery embolism, electroacupuncture treatment can inhibit the pathological inflammatory reaction mediated by NLRP3 inflammatory corpuscles through the pathway mediated by α7 nicotinic acetylcholine receptor, thus alleviating the brain injury induced by IS [22]. This prediction result of GO functional enrichment analysis was consistent with the results of many clinical studies, which indicated that bioinformatics analysis could provide new ideas for the prevention and clinical treatment of IS [23]. Many animal experiments and clinical studies have shown that inflammatory response and immune response played an important role in the early stage after stroke, and affected the prognosis and treatment of stroke [24‐27].

Ten key genes in the pathogenesis of IS were identified by using the STRING software of Cytoscape. Most of these key genes were related to inflammatory response, which was the core link of IS injury. Studies had shown [28] that abnormal expression of inflammation-related genes was the genetic basis of IS, and targeted therapy for these genes may provide a new direction for the prevention and treatment of IS. The occurrence and development of IS is mediated by multiple genes [29]. KEGG pathway analysis showed that multiple DEGs mediated the same information transmission pathway, PPI network results also showed that multiple DEGs were closely related, and miRNA results also showed that multiple key genes could predict common target miRNA. Recent studies had found that miRNAs were involved in the pathological and physiological processes of many diseases, including tumors, immune system, cell proliferation, cardiovascular diseases, and nervous system diseases [30]. Stroke affected the level of miRNA in brain and circulation. MiRNA played a key role in the pathogenesis of stroke and its complications, and participated in the regulation of key metabolism, inflammation, and angiogenesis [31]. Studies have shown that miRNAs played a key role in regulating cell growth, differentiation, progression, and apoptosis, as well as neuron development, hematopoiesis, and repair and remodeling of injured tissues [32]. MiRNA-146a is a strong pro-apoptosis factor [33]. As an important inflammatory microRNA, miR-146a can be found in both immune cells and circulating cells [34]. At the level of translation inhibition, miR-146a can act as a negative regulator of inflammation, mediating IL-1 signaling pathway of IRAK1 [35]. In addition, the animal I/R model study explored the relationship between miR-146a and myocardial I/R injury in mice heart by upregulating the expression of miR-146a, and confirmed that miR-146a can protect myocardium from I/R injury [36]. MiRNAs can mediate the pathological and physiological processes of stroke and its complications. Studies have shown that miRNAs is promising as a treatment for stroke and its complications [37]. Samaraweera et al. [38] found that miR-335 can downregulate the expression level of HAND1 and JAG1, block the growth and differentiation of neurons, and participate in the regulation of neuronal development. Tom et al. [39] found that overexpression of miR-335 can downregulate the expression of AP-1 which is related to cell proliferation, differentiation, and apoptosis. These studies suggest that miR-335 may be an important neuronal regulatory factor, and the abnormal expression of miR-335 may be closely related to nervous system diseases. Our current results indicated that some miRNAs including mir-146a, mir-335, mir-27a, and mir-7 may play a key role in the occurrence and development of IS diseases, and the role of these miRNAs in IS may need further discussion. In addition, the research on stroke genes and miRNAs is still limited.

Gene regulatory network plays an important role in the pathophysiological process of stroke. This discovery will help us better understand the pathogenesis of stroke and provide effective and novel treatment strategies for stroke. However, our research also has some limitations, such as the following: (1) Our current research only involves the top 10 hub genes; (2) the specific molecular mechanism of hub genes and miRNAs in stroke regulation is insufficient; (3) in the constructed network, there is a lack of research on the functions of hub genes and miRNA.

Our findings suggested that compared with the healthy control group, the expressions of CEACAM8, CD19, MMP9, ARG1, CKAP4, CCR7, MGAM, CD79B, and CLEC4D in patients with IS were significantly upregulated, which may have an important influence on the pathophysiological mechanism of ischemic stroke. Some potential target miRNAs such as hsa-mir-146a-5p, hsa-mir-7-5p, hsa-mir-335-5p, and hsa-mir-27a-3p were also predicted. Identification of these genes and miRNAs may contribute to the development of early diagnostic strategies, prognostic markers, and therapeutic targets for IS. However, experimental research is still necessary to validate the functions of these molecules in IS.