Ischemic stroke is one of the most common cerebrovascular diseases, and its mortality rate is increasing year by year. Many stroke survivors are being left with various neurological defects, leading to different degrees of impaired quality of life, and this leads to a great burden on patients, their families, and society (Benjamin et al.,2018). The occurrence and development of ischemic stroke are closely related to genetic changes. The identification of genes that regulate the progress of stroke can promote the understanding of the underlined mechanisms of stroke, predict the progress of stroke, and contribute to the development of new treatments.
In this study, the GSE119121 dataset was retrieved from the GEO database, and 14 modules were obtained after the co-expression module analysis with the dynamic hierarchical tree cutting algorithm. 8 gene modules were identified by WGCNA among the groups of the overexpressed genes. The most prominent of these were the brown module and the black module. GO and KEGG analysis showed that the brown module was mainly enriched in signal mechanism such as innate immune system, regulation of defense response, production of cytokines, inflammatory response, endoplasmic reticulum stress response, and so on, with all of them being highly related to stroke. This is consistent with previous reports. In recent years, the role of inflammasome-mediated inflammatory pathways in various central nervous system diseases has attracted increasing attention (Cribbs et al.,2012). Cheon, Mu et al. reported increased levels of pro-inflammatory cytokines, such as IL-1β or IL-18, in the brains of both animals and stroke patients (Cheon et al.,2018) (Mu et al.,2018). In addition, anti-inflammatory therapy also has a neuroprotective effect on stroke. Then, the GSE16561 dataset was analyzed using the SVM-REF algorithm to identify the most related to the disease genes, and intersect them with the genes of the black and the brown modules, ending up with a final list of four hub genes: DEGS1, HSDL2, ST8SIA4 andSTK3. The four hub genes were verified in the second data set, and all four hub genes were significantly increased in the stroke group (P < 0.01), suggesting that these four genes are risk factors for stroke development.
DEGS1 is a protein-coding gene. Karsaig has identified DEGS1 as a pathogenic gene involved in myelin degeneration in the central nervous system and peripheral nervous system (Kraveka et al.,2007). Previous studies have shown that sphingolipid is increasingly regarded as an important cellular mediator in tumor and inflammatory hypoxia (Riechers et al.,2016). Sphingolipid is also an important part of the cell membrane, which is functionally related to basic processes, such as cell differentiation, neuronal signal transduction, and myelin formation. Sphingolipid imbalance is the chief culprit of various neurological diseases (Karsai et al.,2019). Defects in sphingolipid synthesis or degradation can lead to various nervous system diseases. The high expression of DEGS1 after cerebral ischemia promotes the synthesis of ceramides, which may affect the balance between sphingolipids and dihydrosphingolipids. DEGS1 enzyme participates in catalyzing biosynthesis of ceramide from the head and controls the process from dihydroceramide to ceramide, which is essential for maintaining the balance regulation between sphingolipid and dihydrosphingolipid (Rodriguez et al.,2014). Previous studies have shown that ceramide can be used as a predictor of ischemic stroke (Mohamud et al.,2019). Alsanafi believes that manipulating dihydroceramide levels may represent a new treatment strategy for cancer. This leads to the accumulation of dihydroceramide, which ultimately leads to the activation of endoplasmic reticulum stress and autophagy before cancer cells die (Alsanafi et al.,2020). The polyubiquitin of DEGS1 seems to change its function from promoting apoptosis to survival, which plays a vital role in the mechanism of stroke treatment. The confirmation of this validation could suggest that DEGS1 could also be used as a target for the treatment of stroke.
HSDL2 is peroxisome located in human, mouse, and rat cells, and a potential key regulator of lipid metabolism. HSDL2 can regulate lipid metabolism and participate in cell proliferation and apoptosis. Abnormal lipid changes are the pathological basis of stroke, HSDL2 may be involved in the occurrence and development of stroke by regulating lipid metabolism (Zhang et al.,2018). Recent evidence shows that HSDL2 is highly expressed in breast cancer (Dong et al.,2019), lung adenocarcinoma (Shi et al.,2020), thyroid papillary carcinoma (Zeng et al.,2019), ovarian cancer (Zhang et al.,2019), and human neurogliocytoma (Ruokun et al.,2016). The overexpression of HSDL2 promotes apoptosis, but its mechanism in cerebral infarction should be further studied.
STK3 is a pro-apoptotic kinase in inhibiting growth. This kinase promotes chromatin condensation during apoptosis and has been proved to be the substrate of Caspase-6 in biochemistry. Caspases 3, 6, and 7 are involved in the execution of apoptosis. Casp6 has become an important marker for Huntington's disease, Alzheimer's disease, and cerebral ischemia, and it is activated in the early stages of the disease. After brain injury, CASP6 induces neuronal death by triggering an apoptotic cascade, which in turn affects the recovery of nerve function, and can be used as a potential therapeutic target for neurological diseases (Girling et al.,2018).CASP6 also plays a key role in axonal degeneration, which further pinpoints the importance of this protease in the pathways of neurodegeneration (Riechers et al.,2016).
ST8SIA4, a protein-coding gene, mainly synthesizes PolySia, which is a complex sugar that participates in many processes of nerve development, such as neuroblast migration, outward neurite growth, axon pathfinding, axonal bundle formation, and synaptic formation. PolySia is almost completely attached to the neural cell adhesion molecule (NCAM) in the brain. NCAM is an adhesive glycoprotein located on the cell surface of neurons and immune cells and is involved in many important biological functions, including cell adhesion and migration, synaptic formation, learning and memory consolidation, and social interaction. PolySia-modified NCAM is thought to be related to many specific functions of the brain (Burgess et al.,2008). In addition, polySia can be used as a reservoir of neurological (biological) active molecules, such as neurotrophic proteins (e.g. BDNF) (Kanato et al.,2008), catecholamine neurotransmitters (e.g., dopamine) (Isomura et al.,2011), growth factors (e.g., fibroblast growth factor (FGF2, bFGF) (Ono et al.,2012)).
The differential feature enrichment of the four hub genes was further performed using the GSVA algorithm to study the correlation between the hub gene and the functional characteristics of stroke progression. The results showed that most of the hub genes are involved in the pathological process of stroke through the inflammatory response pathway, the PI3K signal pathway, and the P53 signal pathway, while they were also associated with the prognosis.
The inflammatory response pathway includes processes such as inflammatory cell infiltration, apoptosis, and oxidative stress. Several studies have shown that apoptosis is triggered by stroke. Caspase is a marker protein in the process of cell apoptosis, and important factors include caspase-3, caspase-8, and caspase-9. When subjected to stress, cytochrome C released by the mitochondria binds to procaspase-9 /Apaf-1 to activate and cleaved caspase-9 (Ying et al.,2020). The lytic caspase-9 further copes with other caspase members, initiating the caspase cascade, and then triggering apoptosis (Li et al.,2019). Activated caspase-8 cooperatively cleaves and activates the caspase of downstream effector molecules caspase-1, caspase-3, caspase-6, and caspase-7 and amplifies the apoptosis signals (Buschhaus et al.,2018). Clinically, in the acute phase of cerebral infarction, certain effects, such as reducing secondary brain injury and promoting nerve regeneration, have been achieved by inhibiting the process of apoptosis.
The PI3K/Akt signaling pathway is involved in various cellular processes, and it has been proved that the activation of this pathway is related to the occurrence and development of angiogenesis. The negative regulation of angiogenesis promotes the genes of thrombosis, vascular permeability, and inflammation, protecting thus vascular function (Lugovaya et al.,2020). On the one hand, RelA, NF- κ B/ Rel family plays an important role in the response to inflammation and immunity, which may constitute the regulatory signal of PI3K-AKT, and then regulate the pathway of apoptosis (Gao et al.,2019). On the other hand, several studies have confirmed that drugs can improve stroke symptoms by regulating the PI3K/Akt pathway (Lugovaya et al.,2020). A recent study has also reported that PI3K/Akt regulates apoptosis and that the activation of the PI3K/Akt pathway after stroke plays a protective role in neuronal apoptosis (An et al.,2017).
Under the pathological condition of stroke, p53 plays an important role in the regulation of apoptosis and the cell cycle (Guo et al.,2020). The increase of cyclin chaperone D level will affect the process of cells entering the S phase under the regulation of p53(Saito et al.,2005). The degradation of p53 hinders its role in the regulation of apoptosis (Gao et al.,2020). MDM2 is the ubiquitin ligase of p53 and plays a central role in regulating the stability of p53. Akt mediates the phosphorylation of MDM2 in Ser166 and Ser186 and increases its interaction with p300, so MDM2 mediates the ubiquitination and degradation of p53(Liu et al.,2020). Phosphorylation of MDM2 also blocks its binding to p19ARF and increases the p53 degradation (Wang et al.,2020). It can be seen that many genes are involved in stroke through their interactions.