Circulating extracellular vesicles regulate ELAVL1 by delivering miR-133a-3p which affecting NLRP3 mRNA stability inhibiting PANoptosome formation

doi:10.21203/rs.3.rs-4975982/v1

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Research Article

Circulating extracellular vesicles regulate ELAVL1 by delivering miR-133a-3p which affecting NLRP3 mRNA stability inhibiting PANoptosome formation

https://doi.org/10.21203/rs.3.rs-4975982/v1

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Background

In the quest to elucidate novel therapeutic strategies for myocardial injury, recent investigations have underscored the pivotal roles played by circulating extracellular vesicles (EVs) in intercellular communication.

Method

EVs were extracted from individuals who had experienced AMI-EVs and those who were N-EVs. To assess the impact of circulating EVs on cardiomyocyte and endothelial cell proliferation, apoptosis, migration, and tube formation, a range of in vitro assays such as CCK8, EdU assays, flow cytometry, wound healing assays and angiogenesis assays were conducted. Differentially expressed miRNAs in EVs were validated using microarray analysis and real-time PCR. Through bioinformatics analysis, ELAVL1 was identified as a potential downstream target of miR-133a-3p. This finding was further confirmed by conducting dual-luciferase reporter assay and RNA co-immunoprecipitation experiments. To investigate the regulatory effects of circulating EVs from various sources on myocardial injury and PANoptosis, an animal model of ischemia-reperfusion-induced myocardial injury was established.

Result

Our findings revealed that circulating EVs effectively deliver miR-133a-3p to target cells, where it binds to ELAVL1, leading to a decrease in NLRP3 mRNA stability. This reduction in NLRP3 mRNA stability subsequently inhibits the assembly of the PANoptosome, a multi-protein complex implicated in PANoptosis. As a result, we observed a significant mitigation of PANoptosis in our myocardial injury models, demonstrating the protective role of miR-133a-3p against excessive cell death.

Conclusion

The present study underscores the regulatory role of circulating EV-delivered miR-133a-3p in modulating PANoptosis through ELAVL1-mediated NLRP3 mRNA stabilization. This mechanism represents a potential therapeutic target for attenuating myocardial injury by suppressing PANoptosis.

Extracellular vesicles

ELAVL1

PANoptosis

Myocardial injury

miR-133a-3p

Acute myocardial infarction (AMI) is currently the leading cause of death and disability in humans [1]. Current studies have shown that timely reperfusion therapy (such as percutaneous coronary intervention, coronary artery bypass grafting and early thrombolysis) is the most effective way to save patients with AMI [2]. However, reperfusion therapy is often accompanied by a series of cardiac-related adverse events such as myocardial stunning, reperfusion arrhythmia, and microvascular dysfunction, resulting in further aggravation of myocardial injury, a phenomenon known as myocardial ischemia-reperfusion injury (MIRI) [3].

Extracellular vesicles (EVs) were goblet lipid bilayer membrane vesicles with a diameter of 30–150 nm, which are released by various types of eukaryotic cells into extracellular fluids such as blood, cerebrospinal fluid, saliva, and bile [4]. It constitutes extensive communication networks implicated in the regulation of various processes such as development, immunity, tissue balance, tumors, and neurodegenerative disorders. Thorough investigations have revealed that EVs harbor a significant abundance of miRNAs which can exert crucial regulatory functions in myocardial injury via autocrine and paracrine mechanisms.

MiRNAs are a class of endogenous, non-coding single-stranded small RNAs of about 22 nucleotides in length [5]. They are the main factors regulating gene transcription and are of great significance in exosome-mediated intercellular communication. Exosomes transport and release miRNAs to target cells through plasma membrane fusion, cell pinocytosis, and specific receptor-dependent pathways [4]. The main function of EVs-miRNAs is to participate in the negative regulation at the target gene level. A single miRNA can regulate multiple target gene loci, and a single mRNA can also be regulated by multiple miRNAs [6]. If the miRNAs exhibit complete complementarity with the target mRNAs, degradation of the target mRNAs occurs. Recent investigations have revealed that differential expression of EVs-miRNAs in patients suffering from cardiovascular disease can exert control over transcription and translation processes of diverse genes, thereby intervening in pathophysiological mechanisms such as inflammation, cell proliferation, and apoptosis. Consequently, they are progressively emerging as a pivotal therapeutic target for cardiovascular disease treatment [7].

PANoptosis, not merely a recently acknowledged form of pro-inflammatory programmed cell death, but also an emergent concept that highlights the crosstalk and coordination among three cell death modalities – pyroptosis, apoptosis, and necroptosis[8]. This unique process is driven by a multi-protein complex known as the PANoptosome, which facilitates the crosstalk and regulation between these processes[9]. Recent studies have unveiled that the PANoptosome harbors pivotal molecules essential for pyroptosis, apoptosis, and necroptosis, including GSDMD, Caspase1, Caspase3, Caspase8, RIPK1, and RIPK3, enabling the activation and execution of all three pathways[10]. It is hypothesized that this conceptual complex forms a flexible scaffold capable of recruiting the core components of distinct cell death modes to execute cell demise[11]. Notably, NLRP3 plays a critical role indispensable for the activation of all three pathways[12]. Therefore, PANoptosis can further exacerbate inflammation and induce myocardial cell death, highlighting the significance of inhibiting PANoptosis as a crucial strategy for myocardial protection and mitigation of AMI injury. However, there is still a need for further investigation into the regulatory mechanisms underlying PANoptosis in AMI injury.

Therefore, this study aimed to investigate the therapeutic effects of EVs derived from various sources on myocardial injury. Specifically, we examined how miR-133a-3p modulates the impact of cardiomyocyte PANoptosis on exosome-mediated treatment of myocardial injury through its regulation of ELAVL1.

Clinical samples

The AMI patients included in this study were recruited from the Sichuan Provincial People’s Hospital. The diagnosis of AMI was based on the criteria outlined in the 2017 ESC Guidelines. For the control group, volunteers who visited the hospital for a physical examination during the same time period were selected. There were no significant differences observed in demographic characteristics such as age, gender, and weight between healthy individuals and those with AMI. All case information is presented in Supplementary Table 1. All participants provided informed consent after understanding the study protocol, which had been approved by the Ethics Committee of Sichuan Provincial People’s Hospital.

Isolation of serum EVs

After the samples were collected, they were divided into aliquots of 500µL per tube on ice, and the insufficient part was supplemented to 500µL with 1×PBS. Subsequently, cell debris was removed by centrifugation, and the sample was transferred to a 1.5-ml centrifuge tube and centrifuged at 3000 ×g for 10 minutes at 4 ℃. On further removes impurities to pieces, the centrifugal clear liquid transferred to the new tube, in 4 ℃, and 12000 g under the conditions of the centrifugal for 10 minutes. Next,400 µL of precooled Solution A was added to the 500µL blood sample and immediately mixed through a vortex oscillator for 30 seconds, followed by centrifugation again at 12,000×g for 20 minutes to remove miscellaneous proteins. After that, the supernatant was transferred to a new tube, and 120µL of Solution B was added, vortexed and shaken again for 1 minute, and then left at 4 ℃ for at least 30 minutes. Next, the mixture was removed and centrifuged at 12,000×g for 15 minutes at 4 ℃, and the supernatant was discarded, retaining the exosomes-rich precipitate. Centrifugation was repeated for 2 min to further remove the supernatant. After that, the centrifuged precipitate was resuspended with an appropriate amount of 1×PBS and transferred to a new 1.5mL centrifuge tube. Finally, the centrifuge tube containing the resuspension was centrifuged at 12,000×g for 2 min, retaining the supernatant, which was rich in exosome particles. To purify exosomes, the harvested crude exosomes were transferred to the upper chamber of an ExosomePurification Filter (EPF column) and centrifuged at 3 000×g for 10 minutes at 4℃. The liquid at the bottom of the EPF column was collected as the purified exosomes. The purified exosomes should be packaged and frozen in a low-temperature refrigerator at -80℃ for subsequent experiments.

MiRNA microarray analysis

After the normalization and log2 transformation of the microarray data, limma, an R software package, was used to identify DEGs between the AMI-EVs and N-EVs. The selection criterion for DECs was a p of < 0.05 and a | log2(fold change) | > 0. The Robust rank aggregation package in R was used to integrate and rank all harvested DECs with the criterion of p < 0.05. The RRA method was based on the theory that genes in each experiment were randomly ordered. For the genes ranking higher in the experiment, the possibility of differential expression is inversely proportional to the value of p. R software was used to draw the volcano plot.

Cell culture

The AC16 human cardiomyocytes, HUVEC human umbilical vein endothelial cells, and HEK-293T cells were obtained from a Chinese company called Procell biological. All cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% dual antibiotics (penicillin and streptomycin). The cells were incubated under the conditions of 37 ℃ and 5% CO₂.

Flow Cytometry

The cells were harvested and subjected to the staining protocol provided by the Annexin V-FITC/PI apoptosis detection kit. Flow cytometry was employed to visualize apoptotic events, and subsequently, the rate of apoptosis was determined.

Cell Counting Kit-8 (CCK8)

The cells from each treatment group were transformed into a suspension of individual cells, which were then introduced into a 96-well culture plate at a density of 5 × 10³ cells per well. The plate was incubated in a CO₂ incubator at 37°C for 24 hours, followed by the addition of CCK8 working solution to each well. After placing the plate in the incubator for an additional 4 hours, the optical density was measured using a microplate reader.

Dual-luciferase reporter gene assay

ELAVL1 wild-type (ELAVL1-WT) and mutant (ELAVL1-MUT) plasmids containing miR-133a-3p binding sequences were constructed, and the above plasmids were co-transfected with miR-133a-3p NC and miR-133a-3p mimic, respectively. In HEK293 cells, transfection was carried out according to the manufacturer's requirements of Lipofectamine 3000 transfection reagent. After 48 hours, the cells were collected and lysed. The luciferase activity of each group of cells was detected by the dual-luciferase detection kit, and the detection was carried out according to the requirements of the kit.

Quantitative Real-time PCR (qRT-PCR)

Total RNA was extracted from myocardial tissue and cells using TRIzol reagent (Life Technologies). cDNA synthesis was performed using M-MLV Reverse Transcriptase (Takara). The specific products were quantitatively amplified by PCR using the TransStart Green Q-PCR SuperMix Kit (TransGen). β-actin served as a normalization control. The primer sequences are provided in Supplementary Table 2.

Western blot (WB)

The cells and EVs were lysed using a lysis buffer containing radioimmunoprecipitation, and the supernatant was measured using a BCA protein quantification kit. Subsequently, SDS-PAGE gel electrophoresis was conducted, followed by transferring the proteins to PVDF membranes at a current of 90 mA. Prior to adding primary antibodies CD9 (1:2,000, ab307085, abcam, USA), TSG101 (1:2,000 ab125011, abcam, USA), CD63 (1:1,000 ab315108, abcam, USA), VEGFA (1:1,000 ab46154, abcam, USA), ELAVL1 (1:2000 ab170193, abcam, USA) and GAPDH (1:1000, ab8245, abcam, USA) which were incubated overnight at 4°C., the membrane was blocked with skim milk containing 5%. On the following day, horseradish peroxidase-conjugated secondary antibody goat anti-rabbit IgG (diluted at 1:2,000) was applied to the membrane for an hour at room temperature. Finally, chemi-enhanced luminescence reagents were used for visualizing the proteins which were then analyzed on a Bio-Rad digital imaging system.

EdU assays

After completing the EdU labeling process, the culture medium was removed and replaced with 1 ml of a 4% paraformaldehyde solution for fixation at room temperature for 15 minutes. The fixative was then discarded, and the cells were rinsed three times using 1 ml of washing solution per well. Subsequently, each well was exposed to 1 ml of PBS containing 0.3% TritonX-100 for a duration of 15 minutes at room temperature. Following this step, a volume of 0.5 ml Click Reaction was added to every well and gently agitated to ensure uniform coverage over the samples. The plate was incubated in darkness at room temperature for a period of 30 minutes. After removing the washing solution, each well received an addition of 1 ml of DAPI solution (at a concentration equivalent to that denoted as '1X') and further incubation in darkness at room temperature for approximately ten minutes. Any remaining liquid on the slide surface was dried using absorbent paper before sealing it with an anti-fluorescence quencher compound. Finally, images were observed and collected under a fluorescence microscope.

Angiogenesis assay

Refrigerate the matrigel gel at 4°C overnight until it melts, pre-chill the 24-well plate and pipette tips, add 200 µL per well to the pre-cooled 24-well plate, gently centrifuge at a low speed to eliminate any trapped air bubbles, and incubate at a temperature of 37°C for a duration of 30–60 minutes. Obtain HUVEC cells that have been grouped and treat them with 0.25% trypsin for digestion. Create a suspension of single cells using serum-free medium. Inoculate the cells onto the pre-coated matrigel in each well of a 24-well plate following an even distribution of approximately 1×105/cell per well. After incubation under conditions of 37℃ and saturated humidity with CO₂ concentration maintained at around 5%, observe angiogenesis in each treatment group using a microscope.

Wound assay

HUVECs were cultured in six-well plates at a density of 3×105 cells per well. Once the cells had adhered, a 200 µL pipette tip was used to create scratches on the HUVEC monolayer. Subsequently, the culture medium was replaced with serum-free ECM medium supplemented with H₂O₂ (600 µmol/L) and either PBS, N-EVs (100 µg/mL), or AMI-EVs (100 µg/mL). After incubating for 12 hours, cell migration was evaluated using inverted fluorescence microscopy. The acquired images were analyzed using ImageJ software to quantify the area covered by migrated cells.

RNA immunoprecipitation

RNA Binding Protein Immunoprecipitation Assays were performed on PASMCs using the Magna RIP kit from Millipore (17–700) according to the protocol provided. A ELAVL1 specific antibody from Millipore and rabbit IgG purified antibodies (provided in the kit) have been used. The purified RNA was subjected to RT-PCR using specific primers for the binding site of ELAVL1 motif in 3’UTR region of NLRP3.

Animal model

40 adult SD rats were obtained from Charles River Co., Ltd (Beijing, China) and categorized into two groups: the Sham group (thoracotomy performed without ligation of the left anterior descending coronary artery) and the I/R group. The I/R group underwent 30 minutes of LAD ligation followed by perfusion. In addition, there were two treatment groups: AMI-EVs group (LAD ligated for 30 minutes, followed by perfusion and immediate injection of AMI-EVs via tail vein within 5 minutes of reperfusion) and N-EVs group (LAD ligated for 30 minutes, followed by perfusion with no additional intervention). Each group consisted of 10 rats. The administered dose per rat was 200 µl containing 400 µg of both AMI-EVs and N-EVs. All animal experiments were approved by the Institutional Animal Care and Use Committee of Sichuan Provincial People’s Hospital and were carried out under the guidelines of the NIH Guide for the Care and Use of Laboratory Animals.

Statistical analysis

Statistical analysis was conducted using GraphPad software. The measurement data were presented as mean ± standard deviation (x ± s). Analysis of variance was employed, and pairwise comparisons were performed using the Tukey test. A significance level of P < 0.05 was considered statistically significant.

Characterization of EVs of different origins

Firstly, circulating blood exosomes were extracted from both the control and AMI groups, and the concentration of exosomes and total protein was examined. The results showed no statistically significant difference in EVs between the two groups (Supplementary Fig. 1A-B). Additionally, WB detection of CD63, CD9 and TSG101 indicated that the purity and concentration of exosomes from both sources were similar (Supplementary Fig. 1C).

Regulatory effects of EVs from different sources on cardiomyocytes and endothelial cells

Initially, PKH67 was employed to label non-source EVs for validation of their ability to enter AC16 and HUVEC cells (Fig. 1A). Subsequently, the impact of co-culturing EVs from different sources on cell viability was assessed, revealing that N-EVs significantly enhanced cell proliferation. Conversely, AMI-EVs further suppressed cell proliferation as confirmed by EdU staining (Fig. 1B-C). Moreover, examination of apoptosis in both cell types demonstrated contrasting effects; AMI-EVs notably promoted apoptosis while N-EV inhibited it (Fig. 1D). Finally, evaluation of HUVECs' migration and tube-forming capacity revealed that N-EVs significantly augmented these functions whereas AMI-EVs hindered them (Fig. 1E-F).

Effects of EVs from different sources on cardiac function in a mouse model of acute myocardial infarction

We induced AMI in rats through coronary ligation, followed by intramyocardial injection of AMI-EVs, N-EVs, or PBS into the MI border zone. Left ventricular ejection fraction (LVEF) was utilized to evaluate cardiac function enhancement or maintenance. There were no significant differences in LVEF among the four treatment groups at baseline (Fig. 2A). Over the subsequent three weeks, the control group exhibited a gradual decrease in LVEF, while hearts injected with N-EVs demonstrated higher LVEF compared to those of the Sham or PBS groups. Conversely, AMI-EVs-treated hearts displayed worsened LVEF when compared to PBS-treated control hearts (Fig. 2B). In terms of myocardial injury treatment, we confirmed through histological staining that N-EVs could inhibit myocardial fibrosis; however, AMI-EVs aggravated these phenomena (Fig. 2C). Furthermore, based on our in vitro assays indicating that EVs from different sources could regulate angiogenesis, we assessed the expression of angiogenesis markers (VEGFA, CD34 and COX2) in myocardial tissue. Compared to the Sham and PBS groups, N-EVs treatment promoted angiogenesis while AMI-EVs inhibited their expression levels (Fig. 2D-E).

EVs confer protection against oxidative stress on cardiomyocytes and endothelial cells.

EVs play a crucial role in facilitating the exchange of materials and information between cells. To further validate the potential of EVs in mitigating H₂O₂-induced oxidative stress injury, HUVECs and AC16 (600 µmol/L) were subjected to H₂O₂ treatment and co-cultured with N-EVs. In CCK8 and EdU assays, N-EVs (100 µg/mL) exhibited protective effects on both HUVEC and AC16 against H₂O₂ after 24 hours (Fig. 3A-C). The cytoprotective effect of N-EVs against H₂O₂-induced injury was also confirmed through apoptosis assay (Fig. 3D). Moreover, in the tube formation assay, the total branch length observed for N-EVs-treated HUVECs surpassed that of PBS- or H₂O₂-treated counterparts, indicating that N-EVs effectively safeguard cardiomyocytes and endothelial cells from H₂O₂-induced injury (Fig. 3E-F).

The role of miR-133a-3p in EVs

Current research has demonstrated the pivotal role of miRNAs in orchestrating the regulatory mechanisms of EVs. Consequently, employing microarray analysis, we identified a total of 10 up-regulated miRNAs in N-EVs and observed significant down-regulation of 21 miRNAs (Fig. 4A-B). We selected the three most significantly up-regulated miRNAs (miR-133a-3p, miR-144-3p, and miR-23b-3p) for qRT-PCR validation and observed a significant increase in the expression of miR-133a-3p, suggesting its potential role as a crucial regulatory molecule in cardiomyocyte and endothelial cell proliferation mediated by N-EVs (Fig. 4C).

To further investigate the impact of miR-133a-3p on EV-mediated promotion of cardiomyocyte and endothelial cell proliferation, we treated AC16 and HUVEC cells with PBS, N-EV, or AMI-EV. Subsequent qRT-PCR analysis revealed a significant upregulation of miR-133a-3p expression specifically in the N-EV-treated group, while no notable changes were observed in cells treated with PBS or AMI-EV (Fig. 4D). Importantly, when we subjected residual RNA within EV extracts to RNase A treatment to eliminate any potential interference on our results, there was no alteration detected in the expression levels of miR‑133a‑3p (Fig. 4E). Furthermore, upon disruption of EVs using Triton X‑100 followed by RNase A treatment to degrade RNA encapsulated within EVs, we found that the previously increased expression level of miR‑133a‑3p was reversed (Fig. 4F). Notably, the time-dependent and dose-dependent increase in miR‑133a‑3p expression was observed in recipient AC16 and HUVEC cells exposed to N-EVs (Fig. 4G), providing evidence for its encapsulation within EVs and subsequent transfer from donor to recipient cells.

miR-133a-3p influences the PANoptosis in endothelial cells and cardiomyocytes.

We have observed differential expression of miR-133a-3p in N-EVs. Additionally, to confirm the regulatory impact of miR-133a-3p on endothelial cells and cardiomyocytes, we conducted co-treatments using an inhibitor for miR-133a-3p and N-EVs. The outcomes from CCK8 and EdU assays revealed a significant reversal of the proliferative effect induced by N-EVs when inhibiting the expression of miR-133a-3p (Fig. 5A-B).

To further investigate the regulatory effect of miR-133a-3p on cell, we tested the levels of apoptosis, pyroptosis, and necroptosis in the cells. Our findings revealed a significant rise in their expression levels within EVs when miR-133a-3p was inhibited, indicating a reversal of the observed effect (Fig. 5C-F). Furthermore, our in vivo assays also confirmed that N-EVs could inhibit the level of PANoptosis in myocardial tissue (Supplementary Fig. 2).

MiR-133a-3p regulates PANoptosis by targeting ELAVL1

Currently, several studies have demonstrated the crucial involvement of ELAVL1 in PANoptosis regulation. Moreover, utilizing the starBase database, we identified a targeted regulatory relationship between miR-133a-3p and ELAVL1. Consequently, we conducted further investigations to explore their regulatory interplay. We confirmed the specific binding of miR-133a-3p to ELAVL1 through dual-luciferase reporter and RIP assays (Fig. 6A-B, Supplementary Fig. 3). Furthermore, we validated the regulatory effect of miR-133a-3p on cardiomyocyte proliferation using CCK8 and apoptosis assays, demonstrating that ELAVL1 acts as its target gene (Fig. 6C-D). Similarly, we assessed markers of PANoptosis and observed that miR-133a-3p exerts a regulatory role by modulating the expression of ELAVL1 (Fig. 6E-F).

ELAVL1 influences the formation of the PANoptosome by affecting the stability of NLRP3 mRNA.

To delve deeper into the mechanism by which ELAVL1 regulates PANoptosis, we first established ELAVL1 as an RNA-binding protein (RBP). Utilizing predictions from the starBase database, we identified ELAVL1 as an RBP specifically interacting with NLRP3 mRNA (Fig. 7A). Knocking down ELAVL1, we observed no significant changes in the mRNA expression levels of NLRP3 (Fig. 7B), yet there was a marked effect on the protein expression of NLRP3 (Fig. 7C). This led us to hypothesize that ELAVL1 influences the protein expression of NLRP3 by affecting the stability of its mRNA. Our experiments confirmed this hypothesis (Fig. 7D).

Subsequently, we employed the ATtRACT database to predict three binding sites for ELAVL1 on NLRP3 mRNA (Fig. 7E). Through luciferase reporter assays, we validated these predicted sites, pinpointing site P1 as the critical binding region through which ELAVL1 exerts control over NLRP3 (Figs. 7F-G). Additionally, RIP assays provided concrete evidence of the physical interaction between ELAVL1 protein and NLRP3 mRNA (Fig. 7H). Collectively, these findings demonstrate that ELAVL1 regulates PANoptosis by modulating the stability of NLRP3 mRNA.

In this study, we initially investigated the impact of circulating EVs derived from various sources on myocardial injury and discovered that N-EVs exhibited therapeutic potential in treating ischemia/reperfusion-induced myocardial injury. Furthermore, we confirmed the protective effects of N-EVs against hydrogen peroxide-induced cardiomyocyte and endothelial cell injury, as well as their ability to promote cardiomyocyte proliferation and angiogenesis. These findings partially elucidate the underlying mechanisms responsible for the therapeutic efficacy of N-EVs in myocardial injury. Lastly, through microarray analysis, we identified that N-EVs could suppress cardiomyocyte PANoptosis by targeting ELAVL1 via delivery of miR-133a-3p. These novel insights provide valuable directions for developing innovative treatments for myocardial injury (Fig. 8).

Previous studies have found that circulating EVs play different regulatory roles on cardiomyocyte injury [13]. Recent data demonstrate that exosomes engage in cellular interactions through diverse mechanisms, encompassing (a) ligand-receptor binding-mediated recognition of surface receptors on target cells by exosomes, thereby initiating signal transduction; (b) direct fusion between exosomes and target cells to facilitate intercellular receptor transfer and subsequent signal transduction; (c) fusion of exosomes with target cells leading to the release of internal proteins into the cytoplasm of recipient cells, thereby exerting biological functions; (d) delivery of genetic information to target cells by exosomes via mRNA, microRNAs or other transcription factors [13–15]. Our study shows that circulating exosomes can be taken up by cells and undertake intercellular communication while maintaining membrane structure and internal substances, these data support a possible mechanism for the protective effect of circulating EVs against myocardial injury.

Multiple studies have demonstrated the ability of extracellular vesicles (EVs) to encapsulate and transport miRNAs into cells, thereby facilitating their functional roles. The differential expression of miRNAs within EVs compared to blood underscores the significance of investigating these molecules in EVs. Therefore, this study aimed to employ microarray analysis as a starting point for identifying the distinct expression pattern of miR-133a-3p in circulating EVs derived from various sources, with subsequent experiments validating this discovery. As a specific miRNA, the investigation of miR-133a-3p in exosomes is still in its preliminary stage; however, its potential as a biomarker and intercellular communication mediator has garnered significant attention [1, 16]. Particularly in the realm of myocardial injury, miR-133a-3p has demonstrated anti-apoptotic effects and holds promise as a diagnostic biomarker for myocardial injury while being involved in mechanisms related to myocardial protection. These findings offer novel perspectives and potential targets for further research and treatment of cardiac diseases. Our findings indicate that N-EVs possess the ability to modulate cardiomyocyte proliferation and endothelial cell angiogenesis through the action of miR-133a-3p [17–19]. PANoptosis, an essential form of cell death during MI/R injury, is accompanied by the release of numerous inflammatory factors which contribute to further damage in surrounding tissues [20, 21]. As a pivotal RNA-binding protein, ELAVL1 (also known as HuR) has garnered increasing attention in the field of PANoptosis research. Studies have demonstrated that ELAVL1 exerts regulatory control over the expression of key PANoptosis-related proteins such as caspase-1 and NLRP3, thereby actively participating in the signaling pathway governing pyroptotic processes and exerting profound influence on their initiation and progression [22, 23]. Notably, alterations in ELAVL1 expression have been observed to exhibit a direct correlation with augmented or diminished levels of PANoptosis within specific disease models. Although the precise underlying mechanism by which ELAVL1 modulates PANoptosis remains incompletely elucidated, harnessing its potential as both a therapeutic target and strategy holds significant promise [24–27]. Future investigations will delve deeper into unraveling the intricate workings of ELAVL1 within the context of PANoptosis, potentially unveiling novel applications for targeted intervention against specific diseases. The present study demonstrates that N-EVs effectively suppress the expression of ELAVL1 in MI/R cardiomyocytes, thereby attenuating PANoptosis and offering promising clinical implications for mitigating and preventing MI/R injury in ischemic cardiomyopathy.

Although our study is the first to demonstrate the therapeutic potential of N-EVs in myocardial injury, further investigation revealed the crucial role of miR-133a-3p/ELAVL1 pathway in regulating cardiomyocyte PANoptosis. However, due to insufficient clinical research data, it is imperative for our research group to focus on developing comprehensive clinical studies in the future.

In conclusion, our findings demonstrate the pivotal role of circulating exosomes in facilitating intercellular communication through miR-133a-3p and their ability to regulate PANoptosis by targeting ELAVL1, thereby offering a potential therapeutic strategy for cardiomyocyte injury and fibrotic progression.

AMI

Acute myocardial infarction

MIRI

myocardial ischemia-reperfusion injury

EVs

Exosomes

HUVEC

human umbilical vein endothelial cells

FBS

fetal bovine serum

myocardial infarction

PBS

phosphate-buffered saline

LVEF

Left ventricular ejection fraction.

Ethics approval and consent to participate

All animal experiments were approved by the Animal Care and Use Committee of the Ethical Institution of the Aerospace Center Hospital. The human study was approved by the Ethics Committee of Aerospace Center Hospital. Informed consent was obtained from all patient participants included in the study.

Consent for publication

The authors affirm that human research participants provided informed consent for publication.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

This work was supported by the Sichuan Academy of Medical Sciences · Sichuan Provincial People's Hospital High-tech Project [No.30305030721].

Author contributions

Deliang Wang made contribution to the Manuscript writing；Deliang Wang and Ke Liu provided the study materials；Deliang Wang analyzed and interpreted data；Deliang Wang Collected and assembled the data；Ke Liu made contribution to the conception and design；Deliang Wang and Ke Liu were responsible for the experiment and execution. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Zhu W, Sun L, Zhao P, Liu Y, Zhang J, Zhang Y, Hong Y, Zhu Y, Lu Y, Zhao W, et al. Macrophage migration inhibitory factor facilitates the therapeutic efficacy of mesenchymal stem cells derived exosomes in acute myocardial infarction through upregulating miR-133a-3p. J Nanobiotechnol. 2021;19(1):61.
Zhang CS, Shao K, Liu CW, Li CJ, Yu BT. Hypoxic preconditioning BMSCs-exosomes inhibit cardiomyocyte apoptosis after acute myocardial infarction by upregulating microRNA-24. Eur Rev Med Pharmacol Sci. 2019;23(15):6691–9.
Wang L, Yang C, Chu M, Wang ZW, Xue B. Cdc20 induces the radioresistance of bladder cancer cells by targeting FoxO1 degradation. Cancer Lett. 2021;500:172–81.
Liang C, Liu Y, Xu H, Huang J, Shen Y, Chen F, Luo M. Exosomes of Human Umbilical Cord MSCs Protect Against Hypoxia/Reoxygenation-Induced Pyroptosis of Cardiomyocytes via the miRNA-100-5p/FOXO3/NLRP3 Pathway. Front Bioeng Biotechnol. 2020;8:615850.
Huang L, Yang L, Ding Y, Jiang X, Xia Z, You Z. Human umbilical cord mesenchymal stem cells-derived exosomes transfers microRNA-19a to protect cardiomyocytes from acute myocardial infarction by targeting SOX6. Cell Cycle. 2020;19(3):339–53.
Sun L, Zhu W, Zhao P, Wang Q, Fan B, Zhu Y, Lu Y, Chen Q, Zhang J, Zhang F. Long noncoding RNA UCA1 from hypoxia-conditioned hMSC-derived exosomes: a novel molecular target for cardioprotection through miR-873-5p/XIAP axis. Cell Death Dis. 2020;11(8):696.
Taghdisi SM, Danesh NM, Ramezani M, Emrani AS, Abnous K. A novel electrochemical aptasensor based on Y-shape structure of dual-aptamer-complementary strand conjugate for ultrasensitive detection of myoglobin. Biosens Bioelectron. 2016;80:532–7.
Zheng M, Kanneganti TD. The regulation of the ZBP1-NLRP3 inflammasome and its implications in pyroptosis, apoptosis, and necroptosis (PANoptosis). Immunol Rev. 2020;297(1):26–38.
Samir P, Malireddi RKS, Kanneganti TD. The PANoptosome: A Deadly Protein Complex Driving Pyroptosis, Apoptosis, and Necroptosis (PANoptosis). Front Cell Infect Microbiol. 2020;10:238.
Christgen S, Zheng M, Kesavardhana S, Karki R, Malireddi RKS, Banoth B, Place DE, Briard B, Sharma BR, Tuladhar S, et al. Identification of the PANoptosome: A Molecular Platform Triggering Pyroptosis, Apoptosis, and Necroptosis (PANoptosis). Front Cell Infect Microbiol. 2020;10:237.
Zheng M, Karki R, Vogel P, Kanneganti TD. Caspase-6 Is a Key Regulator of Innate Immunity, Inflammasome Activation, and Host Defense. Cell. 2020;181(3):674–e687613.
Malireddi RKS, Kesavardhana S, Kanneganti TD. ZBP1 and TAK1: Master Regulators of NLRP3 Inflammasome/Pyroptosis, Apoptosis, and Necroptosis (PAN-optosis). Front Cell Infect Microbiol. 2019;9:406.
Cao C, Wang B, Tang J, Zhao J, Guo J, Guo Q, Yue X, Zhang Z, Liu G, Zhang H, et al. Circulating exosomes repair endothelial cell damage by delivering miR-193a-5p. J Cell Mol Med. 2021;25(4):2176–89.
Huang Y, Chen L, Feng Z, Chen W, Yan S, Yang R, Xiao J, Gao J, Zhang D, Ke X. EPC-Derived Exosomal miR-1246 and miR-1290 Regulate Phenotypic Changes of Fibroblasts to Endothelial Cells to Exert Protective Effects on Myocardial Infarction by Targeting ELF5 and SP1. Front Cell Dev Biol. 2021;9:647763.
Liao Z, Chen Y, Duan C, Zhu K, Huang R, Zhao H, Hintze M, Pu Q, Yuan Z, Lv L, et al. Cardiac telocytes inhibit cardiac microvascular endothelial cell apoptosis through exosomal miRNA-21-5p-targeted cdip1 silencing to improve angiogenesis following myocardial infarction. Theranostics. 2021;11(1):268–91.
Yan F, Chen Y, Ye X, Zhang F, Wang S, Zhang L, Luo X. miR-3113-5p, miR-223-3p, miR-133a-3p, and miR-499a-5p are sensitive biomarkers to diagnose sudden cardiac death. Diagn Pathol. 2021;16(1):67.
Kuzmin VS, Ivanova AD, Filatova TS, Pustovit KB, Kobylina AA, Atkinson AJ, Petkova M, Voronkov YI, Abramochkin DV, Dobrzynski H. Micro-RNA 133a-3p induces repolarization abnormalities in atrial myocardium and modulates ventricular electrophysiology affecting I(Ca,L) and Ito currents. Eur J Pharmacol. 2021;908:174369.
Li M, Ding W, Tariq MA, Chang W, Zhang X, Xu W, Hou L, Wang Y, Wang J. A circular transcript of ncx1 gene mediates ischemic myocardial injury by targeting miR-133a-3p. Theranostics. 2018;8(21):5855–69.
Liu N, Zhen Z, Xiong X, Xue Y. Aerobic exercise protects MI heart through miR-133a-3p downregulation of connective tissue growth factor. PLoS ONE. 2024;19(1):e0296430.
Bian Y, Li X, Pang P, Hu XL, Yu ST, Liu YN, Li X, Wang N, Wang JH, Xiao W, et al. Kanglexin, a novel anthraquinone compound, protects against myocardial ischemic injury in mice by suppressing NLRP3 and pyroptosis. Acta Pharmacol Sin. 2020;41(3):319–26.
Chen A, Chen Z, Zhou Y, Wu Y, Xia Y, Lu D, Fan M, Li S, Chen J, Sun A, et al. Rosuvastatin protects against coronary microembolization-induced cardiac injury via inhibiting NLRP3 inflammasome activation. Cell Death Dis. 2021;12(1):78.
Lv J, Hao YN, Wang XP, Lu WH, Xie LY, Niu D. Bone marrow mesenchymal stem cell-derived exosomal miR-30e-5p ameliorates high-glucose induced renal proximal tubular cell pyroptosis by inhibiting ELAVL1. Ren Fail. 2023;45(1):2177082.
Zhu B, He J, Ye X, Pei X, Bai Y, Gao F, Guo L, Yong H, Zhao W. Role of Cisplatin in Inducing Acute Kidney Injury and Pyroptosis in Mice via the Exosome miR-122/ELAVL1 Regulatory Axis. Physiol Res. 2023;72(6):753–65.
Huang T, Ding J, Lin L, Han L, Yu L, Li M. Bioinformatic Identification of the Pyroptosis-Related Transcription Factor-MicroRNA-Target Gene Regulatory Network in Angiotensin II-Induced Cardiac Remodeling and Validation of Key Components. Front Biosci (Landmark Ed). 2023;28(11):293.
Jeyabal P, Thandavarayan RA, Joladarashi D, Suresh Babu S, Krishnamurthy S, Bhimaraj A, Youker KA, Kishore R, Krishnamurthy P. MicroRNA-9 inhibits hyperglycemia-induced pyroptosis in human ventricular cardiomyocytes by targeting ELAVL1. Biochem Biophys Res Commun. 2016;471(4):423–9.
Jiang Y, Ge W, Zhao Y, Wu Y, Huo Y, Pan L, Cao S. [LINC00926 promotes pyroptosis of hypoxia-induced human umbilical vein vascular endothelial cells by recruiting ELAVL1]. Nan Fang Yi Ke Da Xue Xue Bao. 2023;43(5):807–14.
Liu N, Xie L, Xiao P, Chen X, Kong W, Lou Q, Chen F, Lu X. Cardiac fibroblasts secrete exosome microRNA to suppress cardiomyocyte pyroptosis in myocardial ischemia/reperfusion injury. Mol Cell Biochem. 2022;477(4):1249–60.

No competing interests reported.

SuppelmentaryTable1.docx
Supplementary Table 1 Patient and Healthy donor characteristics
Supplementarytable2.docx
Table S2. Primer sequence
SupplementaryFigure1.jpg
Supplementary Figure 1 Characterization of EVs of different origins (A) BCA was used to detect the total protein content of EVs from two different sources. (B) The concentration of EVs secreted in each treatment group. (C) WB was used to detect the expression of EVs markers from different sources.
SupplementaryFigure2.jpg
Supplementary Figure 2 In vivo assays verified the role of PANoptosis in the treatment of myocardial injury by N-EVs. QRT-PCR was used to detect the expression of PANoptosis markers in different treatment groups. * vs. Sham group P<0.05. # vs. PBS group P<0.05.
SupplementaryFigure3.jpg
Supplementary Figure 3 The expression of ELAVL1 in different treatment groups was detected by QRT-PCR and WB.

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Circulating extracellular vesicles regulate ELAVL1 by delivering miR-133a-3p which affecting NLRP3 mRNA stability inhibiting PANoptosome formation

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Version 1

Abstract

Background

Method

Result

Conclusion

Figures

Introduction

Material and methods

Clinical samples

Isolation of serum EVs

MiRNA microarray analysis

Cell culture

Flow Cytometry

Cell Counting Kit-8 (CCK8)

Dual-luciferase reporter gene assay

Quantitative Real-time PCR (qRT-PCR)

Western blot (WB)

EdU assays

Angiogenesis assay

Wound assay

RNA immunoprecipitation

Animal model

Statistical analysis

Result

Characterization of EVs of different origins

Regulatory effects of EVs from different sources on cardiomyocytes and endothelial cells

The role of miR-133a-3p in EVs

MiR-133a-3p regulates PANoptosis by targeting ELAVL1

Discussion

Abbreviations

Declarations

Ethics approval and consent to participate

References

Additional Declarations

Supplementary Files

Status:

Version 1