Exosomal micro ribonucleic acid-154-5p derived from vascular smooth muscle cell mediates intracranial aneurysm phenotype in vitro via myosin light chain kinase targeting

Abstract

Objective

The molecular pathogenesis of intracranial aneurysms remains undefined despite its central role in aneurysmal subarachnoid hemorrhage, a major cause of stroke and shock. Although exosomal micro ribonucleic acids have emerged as key regulators in intracranial aneurysm progression, the specific contributions of micro ribonucleic acid-154-5p remain underexplored.

Methods

Using quantitative reverse transcription polymerase chain reaction, we assessed the micro ribonucleic acid-154-5p expression in intracranial aneurysm specimens. Transmission electron microscopy was used to validate the extracted exosome. Exosomal transfer was confirmed using Cy3-labeled micro ribonucleic acid tracing and quantitative reverse transcription polymerase chain reaction. Functional impacts on vascular smooth muscle cells were evaluated using Cell Counting Kit-8, apoptosis, and 5-ethynyl-2′-deoxyuridine assays. Target genes were identified via ribonucleic acid pull-down and luciferase reporter assays.

Results

Herein, micro ribonucleic acid-154-5p levels were upregulated in intracranial aneurysms. Furthermore, micro ribonucleic acid-154-5p promotes the intracranial aneurysm phenotype in vascular smooth muscle cells. H₂O₂–exposed vascular smooth muscle cells secrete exosomal micro ribonucleic acid-154-5p, which can be transferred to other vascular smooth muscle cells. Exosomal micro ribonucleic acid-154-5p promotes the development of intracranial aneurysms phenotype in vascular smooth muscle cells by targeting myosin light chain kinase.

Conclusions

Exosomal micro ribonucleic acid-154-5p is a novel potentially associated and promising therapeutic target for intracranial aneurysm management. However, the current study is limited to in vitro evidence.

Introduction

Intracranial aneurysms (IAs) are abnormal outpouchings that develop on the walls of intracranial arteries and predominantly occur on the Circle of Willis. ¹ Epidemiological data reveal that IA account for >85% of spontaneous subarachnoid hemorrhage cases and rank as the third most common cerebrovascular disorder, following cerebral thrombosis and hypertensive intracerebral hemorrhage. ² Recent epidemiological studies have indicated a rising annual incidence of IA, with a notable trend toward earlier onset in younger populations. ³ IA rupture leads to aneurysmal subarachnoid hemorrhage, a form of hemorrhagic stroke frequently complicated by early circulatory shock and multiorgan dysfunction, which largely contributes to the high mortality and disability burden associated with this disease. ⁴ The molecular mechanisms of IA development and rupture remain underexplored, necessitating further research to elucidate these pathways.

Exosomes have been confirmed to participate in the pathological development process of various diseases, including liver disease, cancer, and heart failure.^5–8 Exosomes transport bioactive cargos, including proteins, lipids, and regulatory ribonucleic acids (RNAs), to modulate recipient cell behavior.^9,10 Among these components, micro ribonucleic acids (miRNAs) have garnered significant attention owing to their gene-regulatory roles. These 22-nucleotide non-coding RNAs post-transcriptionally silence target messenger ribonucleic acids (mRNAs) through sequence-specific interactions.^11–13 At present, it has been confirmed that miRNA, as the main RNA component, is abundant in exosomes.^14,15 miRNAs carried by plasma exosomes also participate in IA development; however, their specific role and mechanism remain poorly studied. ¹⁶

Our experimental data revealed a significant elevation of miR-154-5p expression in both IA tissues and plasma exosomes in IA patients. miR-154-5p has been characterized as a tumor suppressor in multiple carcinomas. ¹⁷ In vascular dementia, miR-154-5p has also been associated with impaired endothelial progenitor cell function and vascular repair processes; however, its role in IA remains unexplored. ¹⁸ Furthermore, we found that downregulating miR-154-5p attenuated H₂O₂–induced vascular smooth muscle cell (VSMC) dysfunction relevant to aneurysm development. Myosin light chain kinase (MYLK) is an established key biomolecule in many vascular disorders and exerts a protective effect in IA development. ¹⁹ Additionally, H₂O₂–treated VSMCs–derived exosomal miR-154-5p accelerates IA development via direct inhibition of MYLK.

Methods

Human samples

Samples included 8 vascular tissues (4 IA tissues vs. 4 normal superficial temporal artery tissues), and 24 serum samples (12 patients with IA and 12 normal individuals) were collected from the patients at People's Hospital of Yixing City. The study complies with all regulations. Written informed consent was obtained from the patients for the publication of any images, clinical data, and other data included in the manuscript. Publicly available data from the Gene Expression Omnibus (GEO) (Accession: GSE66239; 7 IA tissues and 10 superficial temporal artery tissue) were also used. This study was approved by the Ethics Committee of the People's Hospital of Yixing City (147-01, 11/09/2023) and was conducted in accordance with the Declaration of Helsinki of 1975, as revised in 2024.

Cell lines and cell culture

Human aortic VSMCs (American Type Culture Collection, ATCC, USA; CRL-1999, RRID: CVCL_4009) were aseptically maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA) under standardized atmospheric conditions (37°C, 5% CO₂, 95% humidity). Cell culture was conducted using the previously reported in vitro culture method for IA; VSMCs were exposed to 180 μM of H₂O₂ for 6 h incubated. ²⁰

Oligonucleotides and plasmids

GenePharma (Shanghai, China) synthesized all oligonucleotides, including miR-154-5p mimic, miR-154-5p inhibitor, and negative controls (NCs), which were used as controls. The small interference RNAs (siRNAs) for MYLK were also synthesized from GenePharma and the sequence is 5'-GCCAAGAUGUUGUGAGCAATT-3'. The pcDNA3.1-MYLK overexpression plasmid was constructed via using full-length polymerase chain reaction (PCR) amplicons.

5-ethynyl-2′-deoxyuridine (Edu) assay

The DNA replication activity of VSMCs in different treatment groups was evaluated using an EdU Cell Proliferation Kit (Beyotime, Shanghai, China). We conducted the experiment strictly according to the experimental steps, and a Leica laser scanning confocal microscope was used to scan and photograph the cells.

Cell Counting Kit-8 (CCK-8) assay

Cells were seeded in 96-well plates (100 μL/well containing 3000 cells) and incubated it at 37°C with 5% CO₂ for 24 h, thus allowing cell attachment and stabilization. At different time points, CCK8 reagent was added to well of 96-well plates (10 μL/well) using multi-channel pipettes, and plates were incubated for an additional 2 h. The absorbance at 450 nm were measured by using a microplate reader.

Apoptosis assays

The apoptosis ratio was measured using flow cytometry. The Annexin V-FITC apoptosis detection kit was obtained from BD Biosciences (USA). The percentages of apoptosis were detected using double staining of Annexin V-FITC and propidium iodide. We filtered cells through mesh and acquired the data within using a flow cytometer. The apoptosis was quantified by gating Annexin V+/ propidium iodide − and Annexin V+/ propidium iodide + populations.

Exosome isolation, labeling and identification

Extracellular exosomes were enriched from conditioned media/serum using ExoQuick-TC™ kit (System Biosciences, USA) after media/serum was combined with ExoQuick exosome precipitation reagent. The exosome pellet was resuspended in phosphate-buffered saline (PBS), and the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, USA). For subsequent cell treatment experiments, VSMCs were incubated with exosomes at a final concentration of 20 µg/mL. Transmission electron microscopy (TEM) (Hitachi, Japan) was used to detect the isolated exosomes after dual fixation. Exosomal RNA was extracted using miRNeasy Mini Kit (Qiagen, Germany). PKH67 membrane labeling (Sigma) enabled confocal tracking (Leica TCS SP8). Exosomes were fluorescently labeled through the PKH67 Green Fluorescent (Sigma, USA), and images were captured using a confocal microscope.

Cy3-labeled miR-154-5p

H₂O₂–treated VSMCs cells were transfected with either Cy3-labeled miR-154-5p (GenePharma, China) or non-labeled miR-154-5p. Subsequently, VSMCs cells were incubated with exosomes isolated from the H₂O₂–treated VSMCs cells. Following this, PKH67 and 4',6-diamidino-2-phenylindole (DAPI) staining were conducted to label VSMCs cells, and imaging was conducted via using a confocal microscope.

Luciferase reporter assay

The 3'-UTR regions of MYLK harboring the binding site of miR-154-5p was inserted into pMIR-REPORT plasmid, with mutated constructs serving as controls. VSMCs were transfected with miR-154-5p mimics alongside reporter plasmids. The luciferase activity was quantified using a Dual Luciferase Reporter Assay Kit (Promega, USA).

Quantitative reverse transcription (qRT)–PCR

Total RNA was extracted using the Total RNA Extraction Kit (Solarbio, Beijing, China), and the reverse transcription process was conducted utilizing HiScript RT SuperMix (Vazyme Biotech, Nanjing, China) and MicroRNA Reverse Transcription Kit (Applied Biosystems, CA). Amplification procedures were performed on Quant Gene 9600 Real-time Fluorescence Quantitative PCR Detection System (Bioer, Hangzhou, China) following established protocols. For miR-154-5p expression analysis, primers sourced from RiboBio (Guangzhou, China) were employed, with U6 serving as the normalization control. MYLK quantification utilized GAPDH as the reference gene, with specific primers designed as follows: forward 5'-CCCGAGGTTGTCTGGTTCAAA-3' and reverse 5'-GCAGGTGTACTTGGCATCGT-3'.

Western blot analysis

Protein was isolated using the Total Protein Extraction Kit (KenGen BioTech, Nanjing, China). Proteins were loaded in equivalent quantities onto sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) gels and electrophoresed under standardized conditions. Following separation, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, and the membranes were blocked and subsequently incubated at 4°C overnight with primary antibodies specific to α-SMA (ab7817, 1:1000, Abcam, UK), SM22 (10493-1-AP,1:5000, Proteintech, USA), MMP2 (10373-2-AP, 1:1000, Proteintech, USA), MYLK (21642-1-AP, 1:1000, Proteintech, USA), CD63 (25682-1-AP, 1:1000, Proteintech, USA), calnexin (10427-2-AP, 1:5000, Proteintech, USA), p-MLC (29504-1-AP, 1:1000, Proteintech, USA), and MLC (3672, 1:1000, CST, USA) and TSG101 (28283-1-AP, 1:1000, Proteintech, USA). Subsequently, membranes were treated with horse radish peroxidase (HRP)–conjugated secondary antibodies (ABclonal, Wuhan, China) and detected using chemiluminescence. β-actin (66009-1-Ig, 1:20000, Proteintech, USA) served as a loading control for normalization.

RNA pull-down assay

The biotinylated miR-154-5p was synthesized using Ruibo (Guangzhou, China) and transfected into cells. Following transfection, the cells were incubated with M-280 streptavidin magnetic beads (Invitrogen, USA), using biotinylated mutants and NC for experimental comparison. qRT–PCR was subsequently employed to assess MYLK levels in the captured RNA complexes.

Statistical analyses

The results were presented as mean ± SD and were statistically analyzed using the Statistical Package for the Social Sciences (SPSS) software (version 13.0, IBM, USA). All data were tested for normality using the Shapiro–Wilk test before performing Student's t-test or one-way analysis of variance (ANOVA), and Tukey's multiple comparisons test was used to correct for multiple comparisons. The thresholds were defined as follows: (a) p <0.05 indicated significance and (b) p <0.01 denoted marked significance.

Results

miR-154-5p expression is upregulated in IA

We first detected miR-154-5p expression in 7 IA tissues and 10 superficial temporal artery tissues obtained from the public IA dataset (GEO# GSE66239) and discovered that miR-154-5p was overexpressed in IA (p = 0.0404) (Figure 1(a)). We also confirmed the high expression level of miR-154-5p in IA by analyzing 4 IA tissues and 4 normal superficial temporal artery tissue that we collected (p = 0.0346) (Figure 1(b)). Meanwhile, serum exosomal miR-154-5p in IA patients exhibited higher expression levels than normal individuals (p < 0.001) (Figure 1(c)). This consistency indicates its potential as a biomarker for IA. Meanwhile, previous studies have linked miR-154-5p to vascular dysfunctions, such as suppressing endothelial progenitor cell function, angiogenesis, and vascular repair processes. ¹⁸ Therefore, we further investigated the function and mechanism of miR-154-5p.

Figure 1.

miR-154-5p expression is upregulated in IA. (a) miR-154-5p expression detected using the GEO#GSE66239 dataset. (b) miR-154-5p expression levels were analyzed in 4 IA tissues and 4 normal superficial temporal artery tissues. (c) Serum exosomal miR-154-5p was analyzed in 12 IA patients and 12 normal individuals. Data are expressed as mean ± SD, ^ap < 0.05, ^bp < 0.001.

miR-154-5p: micro ribonucleic acid-154-5p; IA: intracranial aneurysm.

miR-154-5p promotes the development of IA phenotype in VSMCs

We incubated VSMCs with H₂O₂ to establish the IA model in vitro. A marked increase in miR-154-5p levels was found in H₂O₂–exposed VSMCs (Figure 2(a)). Subsequent experiments revealed that miR-154-5p inhibitor significantly suppressed miR-154-5p levels in these cells under H₂O₂ treatment (p < 0.001 and adjusted p < 0.001) (Figure 2(a)). Concurrently, CCK8 assay data revealed suppressed proliferative capacity in H₂O₂–treated VSMCs (p = 0.0391， adjusted p = 0.0344) (Figure 2(b)), and EdU assay revealed that DNA replication activity of H₂O₂–exposed VSMCs was significantly inhibited (p < 0.001 and adjusted p < 0.001) (Figure 2(c)), whereas flow cytometry indicated elevated apoptosis rates of cells (p = 0.0014 and adjusted p = 0.0018) (Figure 2(d)). The protein levels of α-SMA and SM22 (contractile proteins implicated in the downregulation of IA pathogenesis) and p-MLC (the direct downstream product of MYLK activity, reflecting VSMC contractile function) were decreased and MMP2 expression (upregulation is related to the pathogenesis of IA) were increased in the H₂O₂ treatment group (Figure 2(e)). Notably, miR-154-5p inhibitor effectively rescued the H₂O₂ induced changes in cell proliferation; DNA replication; apoptosis; and protein expression of α-SMA, SM22, and MMP2 (Figure 2(b) to (e)). These revealed that miR-154-5p enhanced the development of IA–related phenotypes in VSMCs.

Figure 2.

miR-154-5p promotes the development of IA phenotype in VSMCs. (a) Cellular miR-154-5p levels in VSMCs after treatment with 180 μM H₂O₂ for 6 h or miR-154-5p inhibitor detection using qRT–PCR. Experiments were performed independently in triplicate. (b) The proliferative ability of VSMCs was assessed using CCK-8 assay. Experiments were performed independently in triplicate. (c) The DNA synthesis of VSMCs was detected using EdU assay, 50 μm. Experiments were performed independently in triplicate. (d) The apoptosis rate of VSMCs was assessed using flow cytometry. Experiments were performed independently in triplicate. (e) Western blot analysis identified α-SMA, SM22, p-MLC, MLC, and MMP2 expression changes in VSMCs, β-actin was used as a control. Data are expressed as mean ± SD, ^ap < 0.05, ^bp < 0.01, ^cp < 0.001.

miR-154-5p: micro ribonucleic acid-154-5p; IA: intracranial aneurysm; VSMCs: vascular smooth muscle cells; qRT–PCR: quantitative reverse transcription polymerase chain reaction; CCK-8: Cell Counting Kit-8; EdU: 5-ethynyl-2′-deoxyuridine.

MYLK is a direct target of miR-154-5p

To explore the functional mechanism of miR-154-5p, bioinformatics tools, including TargetScan, miRmap, and miRDB were employed to identify candidate targets. The prediction revealed 250 genes potentially interacting with miR-154-5p (Figure 3(a)). Among these candidates, MYLK emerged as a prioritized target due to its established protective effect against IA development and is established as a core gene involved in VSMC contraction in IA. ¹⁹ We generated MYLK luciferase reporter constructs (Figure 3(b)). Transfection of the miR-154-5p mimic into VSMCs significantly reduced luciferase activity in the wild-type MYLK reporter compared with the mutant control (p = 0.0077) (Figure 3(c)). To validate direct binding between MYLK and miR-154-5p in VSMCs, RNA pull-down assays were employed to prove that MYLK was pulled down by biotinylated miR-154-5p (p < 0.001, adjusted p < 0.001) (Figure 3(d)). Furthermore, miR-154-5p mimic treatment decreased MYLK protein levels in VSMCs (Figure 3(e)).

Figure 3.

MYLK is a direct target of miR-154-5p. (a) Bioinformatics software (TargetScan, miRmap, and miRDB) predict the potential target genes of miR-154-5p. (b) The binding sites of miR-154-5p on the 3′-UTR of MYLK, and target sequences of MYLK-3′ UTR were mutated. (c) Luciferase assay of cells transfected with MYLK-WT or MYLK-MUT reporter together with miR-154-5p. Experiments were performed independently in triplicate. (d) RNA pull-down assays were conducted and proved that MYLK was pulled down by biotinylated miR-154-5p. qRT-qPCR detected the level of miR-154-5p, and GAPDH was used as an internal control. Experiments were performed independently in triplicate. (e) Western blot analysis identified MYLK protein expression changes in VSMCs treated with miR-154-5p mimic. β-actin was used as a control. Data are expressed as mean ± SD, ^ap < 0.05, ^bp < 0.01, ^cp < 0.001.

miR-154-5p: micro ribonucleic acid-154-5p; MYLK: myosin light chain kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; RNA: ribonucleic acid; qRT–PCR: quantitative reverse transcription polymerase chain reaction; VSMCs: vascular smooth muscle cells.

miR-154-5p is enriched in exosomes secreted by H₂O₂–exposed VSMCs and is transferred to VSMCs

Subsequently, we explored the role of exosomal miR-154-5p in IA development. Exosomes were purified from H₂O₂–exposed VSMCs, and TEM confirmed their cup-shaped morphology and diameter of 30–150 nm (Figure 4(a)). Western blotting confirmed the purity of exosomal preparations. Positive exosomal markers CD63 and TSG101 were detected in both exosome and cell lysate samples, whereas the endoplasmic reticulum marker calnexin was restricted to cell lysates and absent in exosomes (Figure 4(b)). To track exosome uptake, PKH67-labeled exosomes (green fluorescence) were co-cultured with normal VSMCs, with DAPI (blue fluorescence) staining nuclei. Confocal microscope revealed cytoplasmic localization of PKH67-labeled exosomes in VSMCs (Figure 4(c)). To validate miR-154-5p encapsulation, exosomes were treated with RNase A (5 μg/mL) alone or combined with Triton X-100 at 37°C for 30 min. We found that RNase A alone did not alter miR-154-5p levels, whereas RNase A and Triton X-100 (a membrane-disrupting agent) caused a marked reduction (p < 0.001 and adjusted p < 0.001) (Figure 4(d)), confirming that extracellular miR-154-5p is vesicle-encapsulated. Transfer of exosomal miR-154-5p to VSMCs was further verified using qRT–PCR, which revealed increased intracellular miR-154-5p levels after exosome treatment (p < 0.001 and adjusted p < 0.001) (Figure 4(e)). For visual confirmation, we transfected H₂O₂–exposed VSMCs with Cy3-labeled miR-154-5p (red fluorescence) and found that its secreted exosomes were internalized by VSMCs, and red fluorescent signals were observed in the Cy3-labeled group (Figure 4(f)). These findings collectively demonstrate that exosomes serve as vehicles for miR-154-5p transfer to VSMCs, elevating its expression in recipient cells.

Figure 4.

miR-154-5p is enriched in exosomes secreted by H₂O₂–exposed VSMCs and transferred to VSMCs. (a) Exosomes purified from the culture supernatant of VSMCs were detected using TEM. Scale bar, 200 nm. (b) Western blot analysis identified CD63, calnexin, and TSG101 protein expression changes. (c) Exosomes purified from the culture supernatant of H₂O₂–exposed VSMCs were labeled by PKH67. VSMCs were co-cultured with these exosomes and observed under a confocal microscope, with the non-exosomes group used as the negative control group. Scale bar, 10 μm. (d) miR-154-5p expression in VSMCs, untreated or treated with RNase A and/or Triton X-100. Experiments were performed independently in triplicate. (e) miR-154-5p levels in VSMCs pre-treated with non-exosomes or indicated exosomes for 24 h were detected by qRT–PCR. Experiments were performed independently in triplicate. (f) Exosomes with Cy3-labeled miR-154-5p were added to VSMCs, fluorescence signals were detected under confocal microscope. miR-154-5p without Cy3-label was used as the negative control. Scale bar, 10 μm. Data are expressed as mean ± SD, ^ap < 0.001.

VSMCs: vascular smooth muscle cells; TEM: transmission electron microscopy; miR-154-5p: micro ribonucleic acid-154-5p; qRT–PCR: quantitative reverse transcription polymerase chain reaction.

Exosomal miR-154-5p promotes the development of IA phenotype in VSMCs by targeting MYLK

To further investigate exosomal miR-154-5p in VSMCs, miR-154-5p inhibitor was transfected into H₂O₂–exposed VSMCs. Exosomal miR-154-5p expression was relatively higher in H₂O₂–exposed VSMCs compared with that in NC-transfected and anti-miR-154-5p-transfected H₂O₂–exposed VSMCs (p < 0.001 and adjusted p < 0.001) (Figure 5(a)). VSMCs were incubated with exosomes secreted by cells from different treatment groups. The levels of miR-154-5p in VSMCs were significantly increased after treatment with exosomes from the H₂O₂–exposed VSMCs group; however, there was no significant change after treatment with exosomes from NC-transfected and anti-miR-154-5p-transfected groups (p = 0.0102 and 0.0244) (Figure 5(b)). In VSMCs cells treated with exosomes from the H₂O₂–exposed VSMCs group, the IA phenotypic progression induced by H₂O₂ is more significant, including suppressed proliferation; impaired DNA replication capacity; downregulated expression of MYLK, α-SMA, p-MLC, and SM22; and elevated apoptosis and MMP2 levels (Figure 5(c) to (f)). In contrast, exosomes from NC-transfected and anti-miR-154-5p-transfected groups failed to achieve these related molecular changes or functional alterations (Figure 5(c) to (f)). To evaluate whether exosomal miR-154-5p facilitates IA phenotypic progression through MYLK regulation, we reintroduced MYLK into VSMCs exposed to exosomes derived from H₂O₂–treated VSMCs (Figure 5(c)). The restoration of MYLK expression via plasmid transfection effectively reversed the acceleration of IA–related phenotypic changes induced by exosomes from H₂O₂–exposed VSMCs (Figure 5(c) to (f)). These findings collectively demonstrate that exosomal miR-154-5p modulates IA pathogenesis by targeting MYLK–dependent pathways.

Figure 5.

Exosomal miR-154-5p promotes the development of IA phenotype in VSMCs by targeting MYLK. (a) Exosomal miR-154-5p levels in VSMCs after treatment with 180 μM H₂O₂ for 6 h or miR-154-5p inhibitor were detected using qRT–PCR. Experiments were performed independently in triplicate. (b) miR-154-5p levels in VSMCs cells pre-treated with non-exosomes or exosomes with different treatment factors. Experiments were performed independently in triplicate. (c) The proliferative ability of VSMCs was assessed using the CCK8 assay. Experiments were performed independently in triplicate. (d) The DNA synthesis of VSMCs was detected using EdU assay, 50 μm. Experiments were performed independently in triplicate. (e) The apoptosis rate of VSMCs was assessed using flow cytometry. Experiments were performed independently in triplicate. (f) Western blot analyses identified MYLK, α-SMA, SM22, p-MLC, MLC, and MMP2 expression changes in VSMCs, with β-actin as the control. Data are expressed as mean ± SD, ^ap < 0.05, ^bp < 0.01, ^cp < 0.001.

IA: intracranial aneurysm; MYLK: myosin light chain kinase; VSMCs: vascular smooth muscle cells; qRT–PCR: quantitative reverse transcription polymerase chain reaction; CCK-8: Cell Counting Kit-8; miR-154-5p: micro ribonucleic acid-154-5p; EdU: 5-ethynyl-2′-deoxyuridine.

Discussion

As the leading cause of subarachnoid hemorrhage (SAH), IAs often further develop and even rupture after occurrence, leading to extremely high mortality rates after rupture.^2,4 IA rupture leads to aneurysmal SAH, a form of hemorrhagic stroke frequently complicated by early circulatory shock and multiorgan dysfunction. However, the etiology of IA progression remains poorly understood. There is an urgently need to clarify the mechanisms of IA to develop noninvasive methods for preventing IA progression. VSMCs play a crucial role in IA phenotypic transformation, characterized by downregulation of contractile proteins (α-SMA, SM22) and upregulation of MMPs.^21–23 Recent clinical evidence has further linked matrix remodeling to aneurysm severity; circulating desmosine—a marker for elastin degradation—may be associated with aneurysm severity in patients with aneurysmal subarachnoid hemorrhage, directly supporting the relevance of VSMC dysfunction and extracellular matrix degradation in clinical outcomes. ²⁴ The phenotypic transformation of VSMCs is considered a key pathogenic mechanism in IA. Exosomes, which carry functional biomolecules that can be transferred to receptor cells and promote their phenotypic transformation, act as messengers of intercellular crosstalk in the local microenvironment, facilitating disease progression.^25,26 For example, breast cancer–derived exosomes induce myofibroblast phenotypes. ²⁷ Hypoxia preconditioned mesenchymal-stem-cell exosomal miR-216a-5p repairs spinal cord injury by altering microglial M1/M2 polarization. ²⁸ Recent studies have confirmed that exosomes participate in IA development; however, their specific roles and mechanisms remain unclear. ¹⁶ In this study, exosomes isolated from VSMCs with IA phenotypic transformation induced by H₂O₂ were found to accelerate H₂O₂–induced IA phenotypic progression in recipient VSMCs.

Certain miRNAs could be transferred to recipient cells via exosomes and exert effects by reducing target gene expression at the post-transcriptional level. ²⁹ For instance, exosomes derived by cancer cells can transfer miR-1247-3p to recipient cells and induce cancer-associated fibroblasts through via B4GALT3 regulation, ultimately leading to lung metastasis. ³⁰ Evidence has suggested that plasma exosome–carried miRNAs also play significant roles in IA development; however, research on this remains limited. ¹⁶ Our study found that the miR-154-5p level is significantly elevated in IA. Compared with normal individuals, IA patients have significantly higher serum exosomal miR-154-5p levels. This consistency indicates its potential as a biomarker in IA. miR-154-5p is the tumor-suppressive miRNA in cutaneous squamous cell carcinoma, glioblastoma, and gastric cancers. ¹⁷ In vascular dementia, miR-154-5p is reportedly related to suppression of endothelial progenitor cell function, angiogenesis, and vascular repair processes. ¹⁸ However, this is the first study to investigate its role in IA. Our findings demonstrate that miR-154-5p promotes the development of the IA phenotype in VSMCs. Moreover, miR-154-5p is enriched in exosomes secreted by H₂O₂–treated VSMCs and can be transferred to other VSMCs. These exosomal miR-154-5p molecules further accelerate H₂O₂–induced IA phenotypic progression in recipient VSMCs.

MYLK is positively correlated with immune infiltration. ³¹ MYLK catalyzes the phosphorylation of myosin light chains and regulates the metastasis of certain malignancies. ³² MYLK encodes a critical kinase essential for myosin activation and subsequent interaction with actin filaments; thus, its deficiency leads to impaired contraction in smooth muscle cells. ³³ Accumulating evidence has suggested that MYLK plays a key role in vascular diseases or angiogenesis and serves as a protective factor in IA progression. ¹⁹ We demonstrated that MYLK is the target gene of miR-154-5p. We also proved that exosomal miR-154-5p promotes the IA phenotypic progression of VSMCs induced by H₂O₂ by inhibiting MYLK. Although this study provides novel insights into the role of the exosomal miR-154-5p/MYLK axis in IA pathogenesis, certain limitations should be acknowledged. First, since in vivo validation was not performed, the current findings are primarily derived from in vitro experiments using H₂O₂–treated human VSMCs. Although this model effectively simulates oxidative stress in IA, it cannot fully recapitulate the complex hemodynamic forces and neurovascular interactions present in living organisms. Consequently, the role of miR-154-5p in IA requires further validation in animal models or clinical studies. Second, the limited sample size reduces the statistical power for subgroup analyses and may not fully represent the heterogeneity of IA patients. These human data are exploratory in nature, and validation in a larger independent cohort is warranted. Additionally, although 250 potential targets were identified bioinformatically, only MYLK was validated in this study. We acknowledge that MYLK is likely only one component of a broader regulatory network, and other pathways (e.g. cytoskeletal dynamics and extracellular matrix remodeling) may also be involved. ³⁴ To further support this, we also examined the levels of MLC and p-MLC, which reflect smooth muscle cell contraction activity.

miR-154-5p post-transcriptionally silences MYLK in VSMCs, disabling its protective function and accelerating the H₂O₂–induced IA phenotype in vitro. Meanwhile, miR-154-5p is propagated via exosomes to adjacent VSMCs, establishing a microenvironment supportive of the cellular IA phenotype in vitro. Understanding the role of exosomes and their molecular biological basis in the cellular processes associated with IA progression can facilitate the identification of novel biomarkers and potential therapeutic approaches for IA.