The etiology of cerebral ischemic stroke (CIS) is complex and diverse. The traditional high-risk factors include hypertension, hyperlipidemia, and diabetes, among others (Du et al., 2021). Recently, there have been increasing reports of CIS caused by malignant tumors (Salazar-Camelo et al., 2021; Wu et al., 2021b). Tumors disrupt organ function and affect the stability of the body’s internal environment. Furthermore, the radiotherapy and chemotherapy drugs and surgery used in tumor treatment also significantly impact the body (Sooklal and Siddiki, 2021). Some retrospective studies have reported a risk of CIS in patients with malignant tumors, such as prostate cancer, head and neck tumors, colorectal cancer, and cervical cancer, and most of these patients have no traditional CIS risk factors (Aspberg et al., 2020; Ciolli et al., 2021), suggesting that the occurrence of CIS in some patients with malignant tumors is unrelated to the pathogenesis of traditional cerebrovascular diseases but is directly or indirectly related to tumors, which is called tumor-related CIS. Lung cancer (LC) is one of the tumors with the highest incidence worldwide, and its death rate is increasing. Even in some developing countries, LC is the malignant tumor most frequently occurring. Studies have confirmed the association between LC patients and increased risk of subsequent stroke (Chen et al., 2011; Anai et al., 2022). However, the detailed and incisive mechanism is still unclear. Therefore, clarifying the common pathogenesis of LC and CIS will be of great significance to the studies on the correlation between CIS and LC by providing them with a new theoretical perspective and actively promoting interdisciplinary research.

Recent studies have found that an uncontrolled cell cycle is a significant cause of unordered cell proliferation and carcinogenesis. Meanwhile, numerous experimental animal models confirmed the abnormal expression of cyclin in neurons after ischemia. Therefore, CCNB2 (CCNB2) was selected as our research focus. CCNB2 belongs to type B cell cycle family protein, which is located on chromosome 15q22, and it binds to cyclin-dependent kinases (CDKs) to regulate their activities (Wu et al., 2021a; Xia T. et al., 2021). Different cyclins have different spatiotemporal specificities at specific stages of the cell cycle (Gupta R. et al., 2021). CCNB2 participates in the G2/M phase transformation in the eukaryotic cell cycle by activating CDKs (Raina et al., 2021). The research of Meyer Da showed that the up-regulation of protein kinase CDK5 is the main reason for neuronal damage during CIS. Restraining abnormal CDK5 during CIS can protect dopamine neurotransmission, and obstruct excitatory toxicity (Meyer et al., 2014). Meanwhile, extensive studies have shown that overexpression of CCNB2 is associated with the development and deterioration of colorectal, lung, and breast cancers (Ma et al., 2021; Moradpoor et al., 2021; Wang et al., 2021; Toolabi et al., 2022). Consequently, CCNB2 can be used as a potential introduction to study the common pathways of the two diseases.

To describe the diversity of cell types in body or disease development and to reveal the functions and mechanisms of various types of cells, the gene expression differences between individual cells need to be explored. Single-cell transcriptome sequencing (scRNA-seq) technology analyzes transcriptome sequencing data at the single-cell level through unbiased, high-throughput, and high-resolution methodology (Ranzoni et al., 2021). Presently, the scRNA-seq method is extensively used in the fields of tumor, immunity, neural development, and animal genetics, among others (Fan et al., 2020). Therefore, scRNA-seq is an effective tool for mining molecular mechanisms.

In summary, the objective of this study is to evaluate the expression and clinicopathological significance of CCNB2 in CIS and LC using high-throughput datasets from public databases and to know preliminarily about the biological mechanism of CCNB2 in CIS through molecular classification and scRNA-seq analyzes.

This study indicated the up-regulation of the CCNB2 mRNA expression in CIS and LC through multilevel evidence. Simultaneously, by analyzing the CCNB2 co-expression genes and scRNA-seq data, the potential biological pathways CCNB2 might involve in were explored, which provides a new perspective for researching the mechanisms of the shared biological pathways between CIS and LC. Our study has the following advantages. First, the study included 103 high-throughput datasets, including 6946 experimental and 5592 control samples, which strongly confirmed the up-regulation of CCNB2 mRNA in CIS and LC through integrated analysis. Additionally, it is of great significance to study LC using tissue samples, because the oncogenesis of LC originates from cytology carcinogenesis. Second, it is the first systematic study of CCNB2 considering the shared biological function between CIS and LC, thus enriching the theoretical content of cyclin family genes. Last, there is still a lack of genome-based and high-throughput data analysis methods to mine the biological mechanism of CCNB2. More importantly, the expression pattern of CCNB2 at the single-cell level in brain organoids and LUAD was explored. Overall, this study comprehensively analyzed the potential biological mechanism of CCNB2 in CIS and LC, which indicated that CCNB2 promotes LC and CIS by regulating the cell cycle, protein kinase activity, and interacting with TOP2A.

Initially, through PPI analysis of crossed high-expression genes in CIS and LC, we found that CCNB2 may be an important gene in the common pathway. Furthermore, using MEGENA analysis, a gene module of CIS including 76 genes was obtained. The function and pathway enrichment analysis of the CCNB2 module genes showed that CCNB2 may participate in the formation of CIS and tissue damage caused by CIS, such as “cell cycle,” “protein kinase activity,” and “glycosphingolipid biosynthesis” through the module gene network. Importantly, previous studies suggested that the change in protein kinase activity was related to the occurrence of CIS (Riebeling et al., 2021). Receptor interacting serine/threonine kinase 3 (RIPK3) was an important member of the serine/threonine protein kinase family and a key regulator of programmed necrosis (Liu et al., 2021). It was found that in the advanced stage of atherosclerosis, about half of the death of macrophages in the plaque was caused by programmed necrosis regulated by RIPK (Zhang et al., 2021). Meanwhile, the expression of RIPK3 in carotid atherosclerotic plaque with necrotic part was significantly higher than that in other tissues (Tian et al., 2016). Mitogen-activated protein kinase activated protein kinase 2 (MK2) was mainly activated by mitogen-activated protein kinase 14 phosphorylation (MPK14) (Zhao et al., 2021). It was also found in vivo experiments that hypercholesterolemic low density lipoprotein receptor (IDLR) (-/-)/MK2(-/-) mice could resist atherosclerosis with reduced accumulation of lipids and macrophages in the aorta (Jagavelu et al., 2007). Meanwhile, the study of Ebrahimian T showed that the activation of MK2 mediates the production of inflammatory mediators and increased blood pressure regulated by angiotensin (Ebrahimian et al., 2011). Simultaneously, MAPKs family played an important role in the process of neuronal apoptosis by participating in the regulation of apoptosis related-genes (Zhou et al., 2021). Whether it was transient or permanent ischemic injury, MAPKs expression level altered significantly, and inhibiting its cascade reaction could improve the cerebral ischemic injury in animal models (Yin et al., 2021). Therefore, the regulation of mitogen-activated protein kinase pathway may be an effective way to inhibit cerebral ischemic neuronal injury and protect neural function. The cell cycle is divided into the G1, S, G2, and M phases, and the development of cells depends on the normal operation of the cell cycle, which is an internal component of cell differentiation and proliferation (Lezaja and Altmeyer, 2021). The cell cycle is mainly regulated by cyclins and CDKs. Cyclins are abnormally expressed in CIS, while CDKs play an important role in CIS injury (Saraireh et al., 2012). Inhibiting the activity of the cell cycle has a significant neuroprotective effect. Trimsit et al. found that the expression of CyclinD1 and CDK4 in the penumbra, after cerebral ischemia, is a key marker to evaluate whether ischemic neurons enter the cell cycle again (Wood and Endicott, 2018). Additionally, Katchanov et al. found that in the rat model of cerebral ischemia-reperfusion, the deletion of endogenous CDKI p16INK4a can lead to the delayed death of striatal neurons (Katchanov et al., 2001), indicating that there is a relationship between the cell cycle and neuronal injury. Concurrently, many studies have shown that the composition and metabolism of glycosphingolipids on the surface of the tumor cell membrane will undergo changes, such as the emergence of new glycosphingolipid antigens and the transformation of glycosphingolipid structure, among others (Yu et al., 2020). The alternation correlates with the occurrence and development of tumors and can be distinguished as a tumor marker (Moghimi et al., 2021). Furthermore, inhibiting the high expression of related glycosphingolipids will have a clear palliative effect on the development of tumors and will help to block the metastatic pathway of tumors. For example, Salk found that the sialylation level of the glycosphingolipid sugar chain of cells increased with high malignancy or high metastatic potential, indicating that sialic acid glycosphingolipid may promote the metastasis and deterioration of tumor cells (Yang et al., 2021). These studies have suggested a potential association between cell cycle activity and the related metabolism between LC and CIS.

Subsequently, TOP2A as a hub gene of the CCNB2 co-expression module in CIS was found to have the highest mutation frequency, including missense mutation, frameshift insertion mutation, and multihit, in LUAD and LUSC. DNA topoisomerase is the general name of enzymes that can catalyze the conversion of DNA topoisomerase (Crewe and Madabhushi, 2021). TOP2A widely exists in eukaryotic and prokaryotic cells and mainly regulates DNA topology by participating in DNA division, repair, recombination, replication, and transcription (Di Felice and Camilloni, 2021). Among them, TOP2A is mainly distributed in the nucleus and participates in the cell cycle pathway, which is closely related to cell proliferation and apoptosis (Tian et al., 2021). By analyzing the effect of the expression level of TOP2A protein on the proliferation and invasion of tumor cells, the researchers found that the expression of TOP2A protein was closely related to the proliferation and invasion of LC cells (Kanne et al., 2021). However, there are few studies on TOP2A in the nervous system. Recently, Thakurela found that small molecule inhibitors of TOP2 can destroy the formation of embryonic stem cells and lead to the down-regulation of genes of embryonic pluripotency (Thakurela et al., 2013). Studies by Lauren F Harkin have shown that TOP2A is involved in regulating the acquisition and maintenance of activated astrocyte stem cell characteristics, which may be mediated by transcription factor Paired Box 6 (PAX6) (Harkin et al., 2016). More importantly, CCNB2 and TOP2A were found specifically over-expressed in GABAergic neurons by scRNA-seq analysis. GABA is an inhibitory neurotransmitter mainly existing in the central nervous system and widely distributed in the brain and spinal cord (Sanchez-Vives et al., 2021). The GABAergic signaling system comprises glutamate decarboxylase (GAD), GABA, GABA transporter, and GABA receptor (GABAR) (Han et al., 2021). In the cerebral cortex, the excitatory signals provided by glutamatergic neurons and the inhibitory signals provided by GABAergic neurons always complement each other to achieve the balance of excitation and inhibition (Bell et al., 2021). Jiang conducted an in-depth study on how the activity of GABAergic neurons affected the rehabilitation of neurological function after stroke (Jiang et al., 2017). The results showed that inhibiting the activity of GABAergic neurons in the striatum could promote the improvement of neurological function and reduce the volume of brain atrophy in post-stroke mice. In addition, these protective effects may be related to the inhibition of GABAergic neuron activity and the promotion of the nutritional factor fibroblast growth factor (FGF) up-regulation in vascular endothelial cells. The above-mentioned studies will help us to further understand the pathological process of CNS injury. Additionally, CCNB2 was found up-regulated on T cells expressing proliferative molecules. Pathway enrichment analysis of upregulated genes on the cluster indicates that CCNB2 promotes LUAD via interleukin 18 (IL-18) signaling pathway and vascular endothelial growth factor A (VEGFA)-VEGFR2 signaling pathway. As a proinflammatory cytokine, IL-18 plays an important role in the occurrence and development of infectious diseases and tumors (Gupta A. et al., 2021). Gu RH found that IL-18 exerts a fine prognosis effect on LC patients (Gu et al., 2020). VEGFA is a protein and specific mitotic agent for endothelial cells (Ling et al., 2021). Combined with receptors, VEGFA can promote the division and proliferation of vascular endothelial cells and increase microvascular permeability (Kajal et al., 2021; Lee et al., 2021). Many studies have confirmed that VEGFA is up-regulated in non-small cell lung cancer (NSCLC) tissues and is also significantly positively correlated with lymph node metastasis of NSCLC (Wei et al., 2021; Xia Y. et al., 2021). At the same time, it is also an effective prognostic factor for NSCLC (Huang Y. S. et al., 2021; Shi et al., 2021). In summary, the above results indicate that CCNB2 can be a potential target for treatment strategies for CNS injury repair as well as LC.

Although we attempted to analyze the relationship between LC and CIS using CCNB2 on multiple levels, the following limitations still existed in this study. First, the clinical specimens of patients were not collected, which hindered the collection of clinical parameters, such as tumor, node, and metastasis (TNM) staging. Second, incomplete information prevented us from performing adequate follow-up of patients. There were also insufficient data in the major gene databases and online analysis websites to analyze the influence of CCNB2 expression on the prognosis of CIS and LC. Additionally, the lack of comparison of CCNB2 expression data between LC-related CIS and LC combined with CIS leads to the deficiency in the persuasion of this study. Overall, our results still need to be further confirmed by experiments in vitro and vivo.

In summary, multiple inferences were provided for the up-regulation of CCNB2 in LC and CIS. The effect of CCNB2 on the common pathway between LC and CIS may indicate a foreground of CCNB2 as a biomarker and therapeutic target for LC-related CIS.

The datasets presented in this study can be found in online repositories. ArrayExpress (https://www.ebi.ac.uk/arrayexpress/), Sequence Read Archive (https://trace.ncbi.nlm.nih.gov/Traces/sra/, SRA), and Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/).

This study was conducted in accordance with the declaration of Helsinki and approved by the Ethics Committee of The First Affiliated Hospital of Guangxi Medical University (protocol number #2015-KY-Guoji-259), China.

M-JL, Z-GH, and GC conceived and designed the present study and revised the manuscript. S-BY analyzed the data and drafted the manuscript. G-SL drafted the manuscript. TW, D-SH, Z-JL, LC, and ZY guided statistical analysis. YY analyzed the data. G-YC, JW, B-TY, R-XX, and L-YL filtered and extracted data. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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