Brain-Derived Exosomal Circular RNAs in Plasma as Diagnostic Biomarkers for Acute Ischemic Stroke

Acute ischemic stroke (AIS), commonly known as stroke, is a serious condition characterized by interrupted blood flow to the brain, resulting in tissue damage and neurological deficits. Early diagnosis is crucial for effective intervention and management, as timely treatment can significantly improve patient outcomes. Therefore, there is an urgent need to develop new methods for early diagnosis of AIS. Multiple studies have shown that bioactive molecules contained in exosomes, especially circular RNAs (circRNAs), can serve as ideal markers for various diseases. However, research on exosomes and their circRNAs in the development and prognosis of AIS is relatively scarce. Therefore, this paper aims to explore the feasibility of using circRNAs in brain-derived exosomes from plasma as biomarkers for AIS.

Research Background

AIS is one of the leading causes of disability and death worldwide. Statistics show that the number of deaths and long-term disabilities caused by acute ischemic stroke each year is quite substantial. The current clinical approach to acute ischemic stroke is to recanalize the occluded blood vessel as soon as possible, especially within 24 hours of onset, as reperfusion at this time can salvage the ischemic penumbra tissue. However, although existing multimodal imaging methods can predict the presence of ischemic penumbra, their application in remote areas is hindered by the dependence on high-end equipment and facilities, making it very important to develop a rapid and widely accessible diagnostic method.

CircRNAs are a class of non-coding RNAs that are widely present, especially in brain tissue. CircRNAs are produced by back-splicing of long RNA transcripts and are known for their high stability after formation. Exosomes in body fluids are widely recognized as ideal biological resources for developing diagnostic biomarkers. Studies have found that exosomes are rich in circRNAs, and these circRNAs have high stability and enrichment in exosomes, indicating their importance in biological functions and applications.

Research Source

This paper was jointly completed by researchers including Xinli Jiang, Rui Zhang, Geng Lu, Yu Zhou, Jianfeng Li, Xinrui Jiang, Shuangshuang Gu, Hongwei Liang, and Jun Wang. The study was published in the Journal of Neuroimmune Pharmacology in 2024.

Research Methods

Sample Collection and Processing

A total of 172 suspected stroke patients and 67 non-stroke controls participated in the study, with some samples used for high-throughput sequencing and the rest for real-time quantitative reverse transcription PCR (qRT-PCR) validation. Samples were collected within 72 hours of symptom onset, and blood samples were collected from all patients immediately upon admission. All patients were diagnosed based on magnetic resonance imaging (MRI) or delayed computed tomography (CT).

Exosome Isolation and Analysis

Using high-throughput sequencing methods, we first identified 358 dysregulated circRNAs (including 23 significantly upregulated and 335 significantly downregulated circRNAs) from brain-derived exosomes in the plasma of stroke patients. Specific brain cell exosomes were isolated from plasma using Streptavidin magnetic beads and biotin-labeled antibodies.

Subsequently, we conducted further morphological and marker protein analysis of the exosomes using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. The results showed that these exosomes were rich in exosome markers CD63 and brain-derived exosome-specific markers L1CAM, GLAST, and TMEM119.

Data Analysis

In Bioinformatics and Statistical analysis, Circexplorer2 was used to analyze and annotate circRNA sequence data, perform quantitative normalization and low-intensity filtering, and screen for significantly differentially expressed circRNAs using EdgeR software.

qRT-PCR Validation

To validate the high-throughput sequencing results, five upregulated circRNAs (including hsa_circ_0007290, hsa_circ_0049637, hsa_circ_0000607, hsa_circ_0004808, and hsa_circ_0000097) were selected for qRT-PCR validation in 172 AIS patients and 67 non-AIS controls. The results showed that the expression levels of these five circRNAs in brain-derived exosomes from the plasma of AIS patients were significantly higher than those in non-AIS controls.

ROC Curve Analysis

Through receiver operating characteristic (ROC) curve analysis, the results showed that the areas under the curve (AUC) for hsa_circ_0007290, hsa_circ_0049637, hsa_circ_0000607, hsa_circ_0004808, and hsa_circ_0000097 were 0.8317, 0.8480, 0.8973, 0.8563, and 0.8772, respectively, indicating high specificity and sensitivity of these circRNAs. When the combination of these five circRNAs was used to distinguish AIS patients from non-AIS controls, the AUC value reached 0.9209, with diagnostic sensitivity and specificity of 95.08 and 80.65, respectively.

Main Results

The study found that these five circRNAs were significantly upregulated in brain-derived exosomes from the plasma of AIS patients, and their expression levels positively correlated with the National Institutes of Health Stroke Scale (NIHSS) and modified Rankin Scale (mRS) scores. This suggests that these circRNAs can serve not only as early diagnostic markers for AIS but also for assessing disease progression.

Conclusion and Significance

This study reveals for the first time that circRNAs (hsa_circ_0007290, hsa_circ_0049637, hsa_circ_0000607, hsa_circ_0004808, and hsa_circ_0000097) in brain-derived exosomes from plasma can serve as ideal diagnostic markers for AIS and may become potential targets for therapeutic interventions. However, as the current data is based on only about 200 participants, more samples are needed to further evaluate and validate these findings.

This study provides new ideas and methods for the rapid diagnosis of AIS, with important scientific value and clinical application prospects. Through further research, it may provide new means for early diagnosis and treatment of AIS, improving patient prognosis.