Upregulation of CCNB2 and Its Perspective Mechanisms in Cerebral Ischemic Stroke and All Subtypes of Lung Cancer: A Comprehensive Study

Abstract

Cyclin B2 (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. In this study, 103 high-throughput datasets related to all subtypes of lung cancer (LC) and cerebral ischemic stroke (CIS) with the data of CCNB2 expression were collected. The analysis of standard mean deviation (SMD) and summary receiver operating characteristic (SROC) reflecting expression status demonstrated significant up-regulation of CCNB2 in LC and CIS (Lung adenocarcinoma: SMD = 1.40, 95%CI [0.98-1.83], SROC = 0.92, 95%CI [0.89-0.94]. Lung squamous cell carcinoma: SMD = 2.56, 95%CI [1.64-3.48]. SROC = 0.97, 95%CI [0.95-0.98]. Lung small cell carcinoma: SMD = 3.01, 95%CI [2.01-4.01]. SROC = 0.98, 95%CI [0.97-0.99]. CIS: SMD = 0.29, 95%CI [0.05-0.53], SROC = 0.68, 95%CI [0.63-0.71]). Simultaneously, protein-protein interaction (PPI) analysis indicated that CCNB2 is the hub molecule of crossed high-expressed genes in CIS and LC. Through Multiscale embedded gene co-expression network analysis (MEGENA), a gene module of CIS including 76 genes was obtained and function enrichment analysis of the CCNB2 module genes implied that CCNB2 may participate in the processes in the formation of CIS and tissue damage caused by CIS, such as "cell cycle," "protein kinase activity," and "glycosphingolipid biosynthesis." Afterward, via single-cell RNA-seq analysis, CCNB2 was found up-regulated on GABAergic neurons in brain organoids as well as T cells expressing proliferative molecules in LUAD. Concurrently, the expression of CCNB2 distributed similarly to TOP2A as a module marker of cell proliferation in cell cluster. These findings can help in the field of the pathogenesis of LC-related CIS and neuron repair after CIS damage.

Keywords: cerebral ischemic stroke; co-expression; cyclin B2; lung cancer; single-cell RNA-seq.

FIGURE 1

Differential expression of cyclin B2 (CCNB2) between lung adenocarcinoma (LUAD) and non-LUAD samples. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.

FIGURE 2

Differential expression of cyclin B2 (CCNB2) between cerebral ischemic stroke (CIS) and lung cancer (LC) samples compared with control group. (A) Box-plot of each dataset included in expression analysis of CCNB2 in lung squamous cell carcinoma (LUSC). (B) Box-plot of each dataset included in expression analysis of CCNB2 in small-cell lung carcinoma (SCLC). (C) Box-plot of each dataset included in expression analysis of CCNB2 in CIS. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.

FIGURE 3

Pooled standard mean deviation (SMD) forest plot for CNNB2 between cerebral ischemic stroke (CIS) and lung cancer (LC) samples compared with control group. (A) standard mean deviation (SMD) forest plot for CNNB2 in lung squamous cell carcinoma (LUSC). (B) SMD forest plot for CNNB2 in lung adenocarcinoma (LUAD). (C) SMD forest plot for CNNB2 in small cell lung cancer (SCLC). (D) SMD forest plot for CNNB2 in CIS.

FIGURE 4

Integration analysis of CNNB2 in stroke, lung cancer (LC) and control samples for in-house and external datasets. (A) Summarized receiver operating characteristic (sROC) curve and likelihood ratio of cyclin B2 (CCNB2) reflected ability of CCNB2 expression in distinguishing experimental samples from control samples in lung adenocarcinoma (LUAD). (B) Summarized receiver operating characteristic (sROC) curve and likelihood ratio of CCNB2 reflected ability of CCNB2 expression in distinguishing experimental samples from control samples in LUSC. (C) Summarized receiver operating characteristic (sROC) curve and likelihood ratio of CCNB2 reflected ability of CCNB2 expression in distinguishing experimental samples from control samples in small cell lung cancer (SCLC). (D) Summarized receiver operating characteristic (sROC) curve and likelihood ratio of CCNB2 reflected ability of CCNB2 expression in distinguishing experimental samples from control samples in cerebral ischemic stroke (CIS).

FIGURE 5

Immunohistochemistry (IHC) staining of cyclin B2 (CCNB2) in lung adenocarcinoma (LUAD) and non-tumor tissues (Antibody CAB009575). (A) Negative IHC staining of CCNB2 in non-tumor tissues; (B) Positive IHC staining of CCNB2 in LUAD and LUSC tissues; (C) Scatter diagram of IHC staining displayed expression analysis of CCNB2; (D) The receiver operator characteristic (ROC) curve of IHC staining displayed expression analysis of CCNB2. *P < 0.05.

FIGURE 6

Enrichment analysis of genes up-regulated on cerebral ischemic stroke (CIS) and lung cancer (LC) and Multiscale Embedded Gene Co-expression Network Analysis (MEGENA) of cyclin B2 (CCNB2) in CIS cohort. (A) The hub genes of intersected differentially expressed genes (DEGs). (B) Heatmap of scale cluster. (C) Result of mean rank using majority vote algorithm. (D) Network diagram of CCNB2 module genes. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis for module genes co expressed with CCNB2.

FIGURE 7

Mutation analysis and clinical significance of cyclin B2 (CCNB2). (A) Mutation analysis of CCNB2 and hub genes of CCNB2 module in lung cancer (LC). (B) The clinic-pathological significance of CCNB2 in LC and cerebral ischemic stroke (CIS). (C) Kaplan-Meier survival curves for overall survival and progression-free survival (PFS) of TCGA-LUSC and TCGA-LUAD grouped by expression of CCNB2. *P < 0.05.

FIGURE 8

Consensus clustering analysis of co expression genes of cyclin B2 (CCNB2) in cerebral ischemic stroke (CIS). (A) Line chart and heat map pointed out k = 2. (B) Heatmap of clustering effect while k = 2. (C) Gene set variation analysis (GSVA) of 2 clusters. (D) 2 clusters showed significant differentially expressed CCNB2 expression (E) 2 clusters showed significant pathway enrichment differences. ****P < 0.0001; **P < 0.01; *P < 0.05.

FIGURE 9

scRNA-seq analysis process of GSE184409 and GSE189357. (A) Uniform manifold approximation and projection (UMAP) distribution of 9 annotated cell types in brain organoids. (B) Distribution of cyclin B2 (CCNB2) expression in cell clusters of brain organoids. (C) Distribution of DNA topoisomerase II alpha (TOP2A) expression in cell clusters of brain organoids. (D) UMAP distribution of 9 annotated cell types in lung adenocarcinoma (LUAD). (E) Distribution of CCNB2 expression in cell clusters of LUAD. (F) Distribution of TOP2A expression in cell clusters of LUAD.

FIGURE 10

Function analysis of cell types. (A) Function analysis of 9 annotated cell types in brain organoids. (B) Function analysis of 9 annotated cell types in lung adenocarcinoma (LUAD).

FIGURE 11

Trajectory analysis of brain organoids cell. (A) The two-dimensional distribution map of state shows the trajectory results of brain organoids cells. (B) Display diagram of cell development timing score calculated according to development trajectory analysis. (C) The two-dimensional trajectory distribution map of cluster. (D) The dynamic changes of the expression level of cyclin B2 (CCNB2) and DNA topoisomerase II alpha (TOP2A) during development.

FIGURE 12

Trajectory analysis of T cells in lung adenocarcinoma (LUAD). (A) The two-dimensional distribution map of state shows the trajectory results of T cells. (B) Display diagram of cell development timing score calculated according to development trajectory analysis. (C) The two-dimensional trajectory distribution map of cluster. (D) The dynamic changes of the expression level of cyclin B2 (CCNB2) and DNA topoisomerase II alpha (TOP2A) during development.