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Integrating Bulk RNA and Single-Cell Sequencing Data Unveils Efferocytosis Patterns and ceRNA Network in Ischemic Stroke

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Abstract

Excessive inflammatory response following ischemic stroke (IS) injury is a key factor affecting the functional recovery of patients. The efferocytic clearance of apoptotic cells within ischemic brain tissue is a critical mechanism for mitigating inflammation, presenting a promising avenue for the treatment of ischemic stroke. However, the cellular and molecular mechanisms underlying efferocytosis in the brain after IS and its impact on brain injury and recovery are poorly understood. This study explored the roles of inflammation and efferocytosis in IS with bioinformatics. Three Gene Expression Omnibus Series (GSE) (GSE137482-3 m, GSE137482-18 m, and GSE30655) were obtained from NCBI (National Center for Biotechnology Information) and GEO (Gene Expression Omnibus). Differentially expressed genes (DEGs) were processed for GSEA (Gene Set Enrichment Analysis), GO (Gene Ontology Functional Enrichment Analysis), and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses. Efferocytosis-related genes were identified from the existing literature, following which the relationship between Differentially Expressed Genes (DEGs) and efferocytosis-related genes was examined. The single-cell dataset GSE174574 was employed to investigate the distinct expression profiles of efferocytosis-related genes. The identified hub genes were verified using the dataset of human brain and peripheral blood sample datasets GSE56267 and GSE122709. The dataset GSE215212 was used to predict competing endogenous RNA (ceRNA) network, and GSE231431 was applied to verify the expression of differential miRNAs. At last, the middle cerebral artery (MCAO) model was established to validate the efferocytosis process and the expression of hub genes. DEGs in two datasets were significantly enriched in pathways involved in inflammatory response and immunoregulation. Based on the least absolute shrinkage and selection operator (LASSO) analyses, we identified hub efferocytosis-related genes (Abca1, C1qc, Ptx3, Irf5, and Pros1) and key transcription factors (Stat5). The scRNA-seq analysis showed that these hub genes were mainly expressed in microglia and macrophages which are the main cells with efferocytosis function in the brain. We then identified miR-125b-5p as a therapeutic target of IS based on the ceRNA network. Finally, we validated the phagocytosis and clearance of dead cells by efferocytosis and the expression of hub gene Abca1 in MCAO mice models.

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Data Availability

No datasets were generated or analysed during the current study.

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Acknowledgements

We acknowledge the original contributors for uploading theirs datasets and the public GEO repository for providing the platform.

Funding

This study was supported by the National Natural Science Foundation of China (82305411), Postdoctoral Fellowship Program of CPSF (2023MD744131), the Innovation Team, and the Talents Cultivation Program of the National Administration of Traditional Chinese Medicine (ZYYCXTD-D-202003), and the Department of Science and Technology of Sichuan Province (2023NSFSC1822, 2021YJ0177).

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  1. Jing Yuan and Yu-sha Liao contributed equally to this work.

Authors and Affiliations

  1. Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu, 610075, Sichuan Province, China

    Jing Yuan, Yu-sha Liao, Tie-chun Zhang, Yu-qi Tang, Pei Yu, Ya-ning Liu, Ding-jun Cai, Shu-guang Yu & Ling Zhao

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Contributions

Ling Zhao is the corresponding author. Jing Yuan and Yu-sha Liao contributed equally to this article. Jing Yuan, Shu-guang Yu, Ding-jun Cai and Ling Zhao designed, processed the data and completed the original draft manuscript; Jing Yuan, Yu-sha Liao, Tie-chun Zhang, Pei Yu, and Ya-ning Liu analyzed and organized the GEO datasets; Yu-qi Tang and Ling Zhao reviewed and revised the manuscript; Jing Yuan and Yu-sha Liao constructed the MCAO model; Shu-guang Yu and Yuan Jing administrated the project and provided the funding.

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Correspondence to Ling Zhao.

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This study was approved by the Committee on Ethical Use of Animals of Chengdu University of Traditional Chinese Medicine (NO. 2023DL-022).

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Yuan, J., Liao, Ys., Zhang, Tc. et al. Integrating Bulk RNA and Single-Cell Sequencing Data Unveils Efferocytosis Patterns and ceRNA Network in Ischemic Stroke. Transl. Stroke Res. (2024). https://doi.org/10.1007/s12975-024-01255-8

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