hsa-miR-518-5p/hsa-miR-3135b Regulates the REL/SOD2 Pathway in Ischemic Cerebral Infarction

You described something but DID NOTHING TO SOLVE STROKE FOR SURVIVORS. Survivors are the reason for stroke research, didn't your mentors and senior researchers tell you that?

hsa-miR-518-5p/hsa-miR-3135b Regulates the REL/SOD2 Pathway in Ischemic Cerebral Infarction

Boyan Zhao and Xiaofan Jiang^*

• Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Xi'an, China

Objectives: Ischemic cerebral infarction (ICI) is a fatal neurovascular disorder. A bioinformatics approach based on single-cell and bulk RNA-seq analyses was applied to investigate the pathways and genes involved in ICI and study the expression profile of these genes.

Methods: First, the aberrantly regulated “small-molecule ribonucleic acids” [microRNA (miRNAs)] and messenger RNAs (mRNAs) were analyzed using transcriptome data from the ischemic brain infarction dataset of the Gene Expression Omnibus (GEO) database. In mouse cerebrovascular monocytes, the single-cell regulatory network inference and clustering (SCENIC) workflow was used to identify key transcription factors (TFs). Then, the two miRNA-TF-mRNA interaction networks were constructed. Moreover, the molecular complex detection (MCODE) extracted the core sub-networks and identified the important TFs within these sub-networks. Finally, whole blood samples were collected for validation of the expression of critical molecules in ICI.

Results: We identified four cell types and 266 regulons in mouse cerebrovascular monocytes using SCENIC analysis. Moreover, 112 differently expressed miRNAs and 3,780 differentially expressed mRNAs were identified. We discovered potential biomarkers in ICI by building a miRNA-TF-mRNA interaction network. The hsa-miR-518-5p/hsa-miR-3135b/REL/SOD2 was found to play a potential role in ICI progression. The expression of REL and superoxide dismutase 2 (SOD2) was significantly elevated in the ICI group in the clinical cohort (P < 0.05). Furthermore, a REL expression was elevated in endothelial cells and fibroblasts at the single-cell level, indicating that REL is a cell-specific regulon. Functional enrichment analyses revealed that REL is primarily engaged in neurotransmitter activity and oxidative phosphorylation.

Conclusions: Our research uncovered novel biomarkers for ICI of neurovascular disease. The hsa-miR-518-5p/hsa-miR-3135b may regulate the REL/SOD2 pathway in ICI progression.

Introduction

Stroke is a common and fatal neurovascular disease that has high morbidity and mortality rates worldwide, accounting for ~17 million new cases annually (1–5). Ischemic stroke (IS) accounts for 80% of all stroke cases (6). Multiple emboli blocking the intracerebral arteries result in irreversible functional deficiencies in local brain tissue, eventually leading to ischemia and hypoxic necrosis (7, 8). Ischemic cerebral infarction (ICI) is a fatal neurovascular disorder (9). Due to its complexity, the molecular pathways underlying the development of ICI are not well-known at the transcriptome level. Exploring the regulatory network of signaling pathways is critical to understanding the mechanism by which ICI develops and to developing effective strategies for preventing and treating ICI.

Single-cell RNA sequencing (scRNA-seq) is a technique for obtaining whole-transcriptome expression profiles at the single-cell level. It is based on the amplification of microscopic whole-transcriptome RNA from isolated individual cells and subsequent high-throughput sequencing to elucidate the molecular regulatory mechanisms underlying specific biological processes and disease pathogenesis (10). In recent years, the scRNA-seq has gradually gained traction in the disciplines of oncology, microbiology, and neuroscience (11–13). In addition to studying changes in gene expression patterns at the population level, the scRNA-seq can be used to study single-cell gene expression, thus, resolving any bias arising due to cellular heterogeneity. Therefore, scRNA-seq is particularly well-suited for studying highly heterogeneous cell populations, such as neural cells (14, 15). Using scRNA-seq, Gate et al. showed that the T-cell receptor (TCR) signaling pathway is activated in CD8+ terminally differentiated effector cells (TEMRA) in the cerebrospinal fluid of patients with Alzheimer's disease, indicating that these cells contribute to the onset of neurological symptoms through their cytotoxic role (16). Vanlandewijck et al. used scRNA-seq to conduct a transcriptional investigation of the constituent cell types of the cerebral vasculature and discovered that endothelial cells, pericytes, and fibroblasts are implicated in the formation of neurovascular disease lesions. In this study, we attempted to address the dearth of molecular studies on cerebrovascular cell types and to establish the groundwork for future research on the molecular pathways underlying cerebrovascular disorders (17). The single-cell regulatory network infeAbegail Floresrence and clustering (SCENIC) is a computational approach for identifying cell states and constructing gene regulatory networks from scRNA-seq data (18). The SCENIC can be used to identify critical transcription factors (TFs) involved in the pathophysiology of ICI.

Apart from single-cell technology, various bioinformatics techniques have emerged as important tools to study complex biological phenomena. The “small-molecule ribonucleic acids” [microRNA (miRNAs)] are a class of non-coding, endogenous single-stranded RNA molecules composed of 20–24 nucleotides that regulate the expression of target genes in various physiological and pathological processes (19). The miRNAs serve as molecular markers for early diagnosis and prognosis, as well as therapeutic targets for ICI (20–23). The miR-PC-5P-12969 inhibits the production of amyloid and promotes IS (24). In acute ICI, the serum miR-124 and other miRs are inhibited, resulting in neuroinflammation and brain damage (25). Additionally, miRNAs can contribute to ICI by modulating TFs. Atherosclerosis (AS) has a significant role in the pathophysiology of ICI (26). Li et al. showed that miR-NA-663 governs the phenotypic metamorphosis of human vascular smooth muscle cells by adversely regulating the expression of its downstream TF, Jun B (27). The miRNA-23b can reduce vascular smooth muscle cell proliferation and migration, and the TF FoxO4 may be a direct target of miRNA-23b (28). Thus, the miRNAs play a critical role in the development of AS by controlling the proliferation, differentiation, and function of vascular smooth muscle cells via TF regulation, potentially altering the course of ICI. Therefore, single-cell sequencing is critical for identifying miRNA-TF-gene regulatory networks involved in ICI progression, which may reveal novel gene targets and molecular markers for ICI diagnosis and therapy.

In this study, we analyzed bulk RNA-seq, as well as scRNA-seq data, to identify a miRNA-TF-mRNA regulatory network that may be vital to ICI progression. Our research aimed to uncover novel biomarkers for ICI of neurovascular disease. The findings of this study may provide new avenues for the prevention and treatment of IS, as well as strategies to improve patient outcomes.

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