Extracellular matrix gene set and microRNA network in intestinal ischemia-reperfusion injury: Insights from RNA sequencing for diagnosis and therapy

Abstract

Intestinal ischemia-reperfusion injury (IIRI) is a complex and severe pathophysiological process characterized by oxidative stress, inflammation, and apoptosis. In recent years, the critical roles of extracellular matrix (ECM) genes and microRNAs (miRNAs) in IIRI have garnered widespread attention. This review aims to systematically summarize the diagnostic and therapeutic potential of ECM gene sets and miRNA regulatory networks in IIRI. First, we review the molecular mechanisms of IIRI, focusing on the dual role of the ECM in tissue injury and repair processes. The expression changes and functions of ECM components such as collagen, elastin, and matrix metalloproteinases during IIRI progression are deeply analyzed. Second, we systematically summarize the regulatory roles of miRNAs in IIRI, particularly the mechanisms and functions of miRNAs such as miR-125b and miR-200a in regulating inflammation, apoptosis, and ECM remodeling. Additionally, this review discusses potential diagnostic biomarkers and treatment strategies based on ECM genes and miRNAs. We extensively evaluate the prospects of miRNA-targeted therapy and ECM component modulation in preventing and treating IIRI, emphasizing the clinical translational potential of these emerging therapies. In conclusion, the diagnostic and therapeutic potential of ECM gene sets and miRNA regulatory networks in IIRI provides new directions for further research, necessitating additional clinical and basic studies to validate and expand these findings for improving clinical outcomes in IIRI patients.

Key Words: Diagnostic biomarkers; Extracellular matrix; Gene expression; Intestinal ischemia-reperfusion injury; Matrix metalloproteinases; MicroRNA; Treatment strategies

Core Tip: This review systematically summarizes the diagnostic and therapeutic potential of extracellular matrix (ECM) gene sets and microRNA (miRNA) regulatory networks in intestinal ischemia-reperfusion injury (IIRI). We review the critical roles of the ECM in the pathological processes of IIRI and discuss the mechanisms by which specific miRNAs regulate inflammation, apoptosis, and ECM remodeling. Based on these molecular insights, potential diagnostic biomarkers and treatment strategies, including miRNA-targeted therapy and ECM component modulation, demonstrate significant clinical translational prospects. In summary, the roles of ECM genes and miRNAs in IIRI provide new research directions aimed at improving patient diagnosis and treatment outcomes.

INTRODUCTION

Intestinal ischemia-reperfusion injury (IIRI) is defined as a severe condition in clinical surgical settings, where local tissue damage during ischemia worsens upon blood reperfusion, leading to systemic inflammatory response and multiple organ dysfunction syndrome (MODS)[1]. This injury results from complex interactions between ischemic tissue damage and subsequent inflammatory responses upon reperfusion, exacerbating tissue damage[2,3]. IIRI is associated with significant morbidity and mortality, particularly in emergency surgeries. Delayed diagnosis and treatment may lead to severe clinical outcomes such as intestinal infarction, sepsis, and death[4]. Factors such as intestinal microbiota, Toll-like receptors (TLRs), oxidative stress, and nitric oxide play crucial roles in the development of IIRI, underscoring the complexity and clinical significance of understanding these mechanisms for improved diagnosis and treatment[2]. Recent studies have demonstrated the potential of various treatment methods in mitigating the effects of IIRI.

IIRI involves multifaceted molecular mechanisms, where oxidative stress, inflammation, and cellular dysfunction play key roles. Ischemia induces vascular constriction and oxidative stress, while reperfusion exacerbates damage through overproduction of reactive oxygen species (ROS), amplifying inflammatory responses[3,5-7]. Emerging evidence underscores the critical role of the extracellular matrix (ECM) gene set and microRNA (miRNA) networks in the molecular mechanisms of IIRI[8]. RNA sequencing (RNA-seq) analyses have revealed dynamic changes in these molecules throughout disease progression, providing valuable insights into the pathways of tissue injury and laying a foundation for the development of innovative therapeutic strategies[9]. Accordingly, further in-depth investigation of ECM gene set and miRNA regulatory networks promises not only to illuminate the molecular basis of IIRI but also to propel advancements in diagnostic and therapeutic methodologies[8].

Based on these advancements, this review examines the potential applications of ECM gene set and miRNA regulatory networks in the diagnosis and treatment of IIRI, highlighting their molecular mechanisms and clinical significance. Gene expression profiling and RNA-seq studies have identified distinct expression patterns of ECM-related genes at various stages of IIRI, which serve as molecular markers for accurate diagnosis and tools for predicting and monitoring disease progression[8]. Non-coding RNAs (ncRNAs), particularly miRNAs and long ncRNAs (lncRNAs), play essential roles in regulating mitochondrial function, apoptosis pathways, and signal transduction[10,11]. For instance, miRNAs such as miR-125b and miR-200a have been identified as promising therapeutic targets, with their modulation shown to significantly mitigate tissue damage and improve clinical outcomes[12]. Additionally, research into competitive endogenous RNA networks highlights the complex interactions among lncRNAs, miRNAs, and mRNAs, underscoring their critical role in regulating gene expression in IIRI[10-12].

RNA-seq analysis has further elucidated the regulatory networks involving miRNAs and ECM genes during the progression of IIRI, offering valuable insights for the development of novel therapeutic strategies[8]. By synthesizing existing research, this review proposes innovative diagnostic and therapeutic approaches centered on ECM gene set and miRNA networks, while also exploring their potential for early disease detection and intervention. These findings deepen our understanding of the complex pathological mechanisms underlying IIRI and provide a foundation for advancing clinical practice with innovative solutions.

PATHOPHYSIOLOGY OF IIRI

Mechanisms of IIRI

IIRI is typically divided into two stages: The first stage is the ischemic phase, during which blood supply disruption causes damage to metabolically active tissues. The second stage is reperfusion, during which blood flow is restored to ischemic tissues[13]. It is recognized that the damage inflicted during the reperfusion phase is often more severe than that during the ischemic phase[14]. During the initial ischemic phase, blood flow interruption leads to intestinal hypoxia, cellular energy depletion, and accumulation of metabolic waste products[13,15]. This hypoxic environment triggers a series of cellular responses, including anaerobic metabolism, lactate accumulation, and alterations in cellular homeostasis. Additionally, intestinal ischemia initiates biochemical events such as ROS formation in affected organs, release of metal ions, and damage to capillary structures. It is accompanied by inflammation, complement activation, and neutrophil infiltration at the site of injury[16]. Oxygen and nutrient deficiency results in cellular injury, increased intestinal barrier permeability, and activation of protease systems. These factors collectively exacerbate the degradation of structural proteins, further compromising cellular integrity[2].

The reperfusion phase occurs when blood flow is restored to the ischemic intestine, paradoxically exacerbating tissue damage. Sudden influx of oxygen leads to ROS generation, causing oxidative stress and damaging cellular components such as lipids, proteins, and DNA[17,18]. Reperfusion also triggers an inflammatory response characterized by endothelial cell activation, leukocyte adhesion, and release of pro-inflammatory cytokines[19,20]. These events lead to increased vascular permeability, edema, and further tissue damage[19,20]. During reperfusion, the intestinal mucosa produces various acute-phase proteins, intestinal hormones, and cytokines[16], contributing to additional mucosal injury[21]. The combination of oxidative stress and inflammatory responses leads to the so-called IIRI, which affects not only the initially affected tissues but may also trigger systemic inflammatory response syndrome[22,23]. Clearly, better understanding of the relevant pathophysiological events of IIRI and early recognition can provide a basis for disease treatment.

Role of ECM in IIRI

The ECM provides structural and functional support to intestinal tissues, maintaining cellular homeostasis and tissue integrity. This dynamic network comprises proteins such as collagen, elastin, fibronectin, laminin, and proteoglycans, which collectively form a scaffold that supports cells, facilitates signaling, and regulates tissue repair[2]. In IIRI, ECM undergoes significant changes due to hypoxia, oxidative stress, and inflammation, disrupting its structural and functional roles[24].

Dynamic changes in ECM components during IIRI: During ischemia, the cessation of blood flow leads to hypoxia and nutrient deprivation, which impair ECM synthesis and turnover. This results in reduced collagen deposition, compromised cross-linking, and disrupted elastin fiber formation, weakening the structural integrity of the intestinal wall[25]. Reperfusion further exacerbates ECM degradation by activating matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, which degrade essential ECM components, including collagen, fibronectin, and laminin[5]. These alterations disrupt cell-ECM interactions, leading to the loss of tissue integrity, increased vascular permeability, and enhanced susceptibility to injury.

ECM degradation and inflammatory modulation: Degradation of ECM components release bioactive fragments, known as matrikines or damage-associated molecular patterns, which amplify inflammatory responses during IIRI[26]. For instance, collagen-derived fragments can bind to pattern recognition receptors (e.g., TLRs) on immune cells, activating signaling pathways that promote cytokine production and leukocyte recruitment[2,27]. This inflammatory cascade not only exacerbates tissue damage but also delays tissue recovery by impairing the reparative processes.

Impact on cellular functions and repair mechanisms: ECM remodeling during IIRI alters cell adhesion, migration, and proliferation, which are critical for tissue repair. The degradation of fibronectin and laminin disrupts integrin-mediated cell anchorage, impairing epithelial and endothelial cell migration required for wound healing[28,29]. Additionally, elastin degradation reduces vascular elasticity, contributing to microvascular dysfunction and further limiting tissue perfusion[30]. These alterations hinder the regeneration of damaged tissues, emphasizing the need to stabilize ECM integrity as a therapeutic approach.

Therapeutic targeting of ECM remodeling: Emerging studies have identified ECM remodeling as a viable therapeutic target for mitigating IIRI-induced tissue damage[31]. Inhibitors of MMPs, such as doxycycline and specific MMP-9 antagonists, have shown promise in preclinical models by reducing ECM degradation and preserving tissue structure[32,33]. Additionally, ECM-mimetic scaffolds and hydrogels, designed to provide structural support and promote cell adhesion, have been explored to facilitate tissue repair and regeneration[34,35]. These biomaterials often incorporate bioactive molecules, such as growth factors and anti-inflammatory agents, to further enhance their reparative potential. Targeting ECM signaling pathways, including integrin and TGF-β signaling, has also demonstrated therapeutic efficacy by modulating the balance between ECM degradation and synthesis[36].

RESEARCH TRENDS IN DIAGNOSTIC METHODS FOR IIRI

IIRI is a prevalent clinical event associated with significant morbidity and mortality[37]. It can arise from conditions such as acute mesenteric ischemia, small bowel transplantation, abdominal aortic aneurysm, hemorrhagic shock, and necrotizing enterocolitis in neonates, as well as in patients with septic shock or severe burns[38,39]. IIRI induces acute vascular dysfunction, disrupts intestinal barrier integrity, and triggers systemic inflammatory responses that can culminate in MODS, with mortality rates as high as 80% in severe cases[40]. Recent studies have shifted focus toward understanding the role of ECM components in IIRI. ECM proteins, such as collagen and laminin, are not only critical for maintaining tissue structure but also mediate repair processes through dynamic remodeling during ischemia-reperfusion. For example, MMPs facilitate tissue repair by modulating ECM degradation and promoting cell migration, although their dysregulation can exacerbate tissue damage[41,42]. Targeting ECM components and their regulatory pathways has emerged as a promising strategy for mitigating IIRI-induced injury and enhancing tissue repair.

Due to the unique anatomical structure of the intestines, they are among the most vulnerable organs to IIRI[43]. First, during severe trauma, major surgeries, and shock, blood flow to the intestines is initially diverted to ensure adequate supply to vital organs like the heart and brain. Moreover, the rate of blood flow recovery to the intestines significantly lags behind that of other tissues and organs. Second, intestinal mucosal blood vessels have a curved circular structure with "shunting" between arterioles and venules, and the high metabolic activity of intestinal villous epithelial cells makes intestinal tissue particularly sensitive to local ischemia. Third, adrenaline receptors are abundant in intestinal mucosal micro-vessels. In severe conditions such as infection, hemorrhage, and trauma, stimulation of adrenaline receptors causes constriction of intestinal capillaries, leading to intestinal ischemia. These structural characteristics make the intestines the initiating organ for systemic inflammatory response and multi-organ dysfunction[21]. IIRI disrupts the integrity of the intestinal mucosa and increases the permeability of the intestinal mucosal barrier. Bacteria and endotoxins enter the bloodstream from the intestine, activating the vascular endothelial system to generate a series of reactions, such as activating macrophages and lymphocytes to release large amounts of cytokines and inflammatory mediators, which amplify damage to distant organs and tissues through cascade reactions[44]. However, the lack of accurate and feasible early diagnostic methods often leads to delays in diagnosis and treatment. For instance, some cases of non-obstructive mesenteric ischemia lack typical clinical symptoms such as abdominal pain, and by the time symptoms of abdominal pain appear, full-thickness intestinal necrosis has already occurred.

Currently, suspected intestinal ischemia is mainly confirmed through selective angiography or exploratory laparotomy[45,46]. However, since many radiological signs of non-obstructive mesenteric ischemia are subtle or absent, arterial computed tomography imaging cannot reliably exclude the presence of intestinal ischemia[47]. Moreover, mesenteric angiography is not only expensive and time-consuming but also unhelpful in diagnosing other acute abdominal conditions, and importantly, it is difficult to apply in critical care medicine (such examinations are difficult to perform on mechanically ventilated and hemodynamically unstable critically ill patients). Although many medical centers are attempting to use other diagnostic methods and devices (such as tension measurement and superconducting quantum interference devices) to diagnose intestinal ischemic injury, further research and exploration are needed due to limited experience in their use and the necessity for large-scale clinical validation. Lin Zhang et al[48] targeted patients with heat stroke-induced intestinal ischemic injury, collected serum from heat stroke patients, and studied intestinal markers such as intestinal fatty acid-binding protein (IFABP) and diamine oxidase (DAO). The results showed significant increases in IFABP and DAO in heat stroke patients, positively correlated with endotoxin levels in the body, possibly due to damage to the intestinal epithelial barrier[49]. At the same time, the study found that serum levels of IFABP, ischemia-modified albumin (IMA), and glucagon-like peptide-1 (GLP-1) were significantly elevated in patients with sepsis complicated by acute intestinal ischemic injury, serving as sensitive and relatively specific serological indicators for early diagnosis. Serum levels of IFABP, IMA, and GLP-1 in septic patients were positively correlated with the Acute Physiology and Chronic Health Evaluation (APACHE II) score. Derikx et al[50] proposed that D-dimer, IFABP, and α-GST are the most promising biomarkers. Treskes et al[21] calculated the joint area under the ROC curve (AUC) of IFABP, D-lactate, α-GST, and D-dimer and found it to be more accurate, with AUC values of 0.876, 0.84, and 0.814, respectively. Overall, based on current research, IFABP, α-GST, D-dimer, and D-lactate appear to be diagnostic markers for acute intestinal ischemia[51], although these markers lack sufficient performance for standalone use. However, further large-sample studies are needed to confirm the exact effectiveness of these markers. Therefore, a rapid, accurate, safe, and minimally invasive diagnostic method is urgently needed in clinical practice.

POTENTIAL OF ECM GENE SETS IN IIRI DIAGNOSIS

ECM gene sets as biomarkers

Definition and importance of gene sets: ECM genes comprises a group of genes that encode ECM proteins, forming a complex network crucial for tissue integrity, cell signaling, and structural support. These gene sets serve as significant biomarkers, reflecting changes in tissue structure and cell interactions during disease processes, including IIRI[52]. Identification of specific ECM genes involved in IIRI contributes to understanding mechanisms of tissue damage and repair, thereby facilitating the development of diagnostic tools and treatment strategies[53].

Specific ECM genes involved in IIRI: Analyzing the differential expression of ECM-related genes using RNA-seq technology reveals key genes and their potential diagnostic value in IIRI. Recent studies have identified genes such as FUNDC1, VDAC1, and MFN2 as critical in hepatic IIRI, where their abnormal expression correlates significantly with the severity of tissue damage[54]. Specific ECM genes, including those encoding collagen, fibronectin, and MMPs, play essential roles in maintaining ECM integrity and mediating tissue remodeling during and after ischemic events[55]. The expression levels of these ECM genes can serve as biomarkers to assess tissue damage severity and evaluate the efficacy of therapeutic interventions in alleviating IIRI[56].

Our current research, complemented by bioinformatics analysis, has identified significantly elevated expression of eight ECM-related genes-COL1A2, THY1, IL10, MMP2, SERPINH1, COL3A1, COL14A1, and P4HA1-in patients with IIRI. These findings are consistent with previous studies, such as that of Jiang et al[57], which employed Cytoscape to construct regulatory networks between miRNAs and mRNAs, identifying COL1A2, THY1, IL10, MMP2, SERPINH1, COL3A1, COL14A1, and P4HA1 as key mRNAs within protein-protein interaction networks. Their research further elucidated miRNA-mRNA regulatory networks in IIRI, highlighting the roles of these mRNAs and their potential functional implications (Table 1).

Table 1 RNA sequencing exploration of RNA-regulated extracellular matrix gene sets.

Gene	miRNA regulation	miRNA regulation
COL1A2	miR-29 family (miR-29a/b/c) targets COL1A2 mRNA, inhibiting its expression	COL1A2 mRNA stability and translation are regulated by RNA-binding proteins such as HuR and TIA-1/TIAR
THY1	miR-21 and miR-199a suppress THY1 expression	THY1 mRNA expression is regulated by various extracellular matrix factors (e.g., TGF-β and FGF-2) and their receptors
IL10	miR-106b and miR-155 reduce IL10 expression by targeting IL10 mRNA	IL10 expression is regulated by inflammation-related signaling pathways (e.g., NF-κB and STAT3)
MMP2	miR-21, miR-221/222, and other miRNAs regulate MMP2 expression by targeting MMP2 mRNA	MMP2 expression is regulated by various extracellular matrix molecules (e.g., ECM components) and their receptors
SERPINH1	miR-29 family (miR-29a/b/c) targets SERPINH1 mRNA, inhibiting its expression	SERPINH1 expression is regulated by extracellular matrix components (e.g., collagen) and related signaling pathways
COL3A1	miR-29 family (miR-29a/b/c) regulates COL3A1 expression by targeting its mRNA	COL3A1 expression is regulated by extracellular matrix components and related signaling pathways
COL14A1	miR-29 family (miR-29a/b/c) affects COL14A1 expression by targeting its mRNA	COL14A1 expression is regulated by extracellular matrix components and related signaling pathways
P4HA1	miR-124, miR-29 family (miR-29a/b/c), and other miRNAs regulate P4HA1 mRNA expression levels	P4HA1 expression is regulated by oxygen concentration and extracellular matrix environment (e.g., collagen content)

miRNA: MicroRNA; MMP2: Matrix metalloproteinase 2; ECM: Extracellular matrix.

Clinical applications of RNA-seq technology

Identifying specific ECM gene sets as biomarkers holds significant potential for early diagnosis of IIRI[58,59]. Monitoring changes in the expression levels of these genes enables clinicians to identify patients at risk of developing severe complications and intervene promptly to prevent further tissue damage[8]. Early diagnosis using ECM gene sets can also guide treatment decisions, allowing for personalized therapeutic strategies targeting the underlying mechanisms of IIRI[10].

The prognostic value of ECM gene sets lies in their ability to predict outcomes and recovery possibilities in IIRI. Changes in ECM gene expression provide insights into the extent of tissue remodeling and the effectiveness of therapeutic interventions, aiding in identifying patients who may require more aggressive treatment[56]. Over time, monitoring ECM gene expression can also help track the progression of IIRI and treatment responses, providing a dynamic tool for managing patient care and improving long-term outcomes[53].

ROLE OF MIRNA REGULATORY NETWORKS IN IIRI

Overview of miRNA functions

miRNAs are a class of small ncRNA molecules that regulate gene expression by binding to complementary sequences on target mRNAs, leading to mRNA degradation or translational inhibition[60]. The biogenesis of miRNAs involves RNA polymerase II transcription, initial processing by the Drosha-DGCR8 complex in the nucleus, and further processing by Dicer in the cytoplasm to generate mature miRNAs[61,62]. These mature miRNAs are integrated into the RNA-induced silencing complex, guiding the complex to target mRNAs based on sequence complementarity, thereby regulating gene expression in various physiological and pathological processes[63].

miRNAs play a crucial role in regulating cellular responses to stress, including IIRI. They modulate gene expression associated with apoptosis, inflammation, oxidative stress, and angiogenesis, influencing cell survival and tissue repair mechanisms[64]. Under ischemic conditions, specific miRNAs are either upregulated or downregulated to orchestrate adaptive responses, such as enhancing antioxidant defenses and attenuating inflammatory damage. These regulatory networks are pivotal for cellular adaptation and recovery from ischemic injury[65].

Specific miRNAs involved in IIRI

RNA-seq studies have identified multiple miRNAs with significant expression changes during IIRI processes (Table 2). For instance, miR-125b and miR-200a are associated with myocardial protection and tissue repair during reperfusion[12]. Other key miRNAs include miR-155, which regulates inflammatory responses, and miR-489, which protects against ischemic injury by targeting genes involved in cellular stress responses[63,66].

Table 2 MicroRNAs exerting effects in intestinal ischemia-reperfusion injury.

miRNA	Meaning	Target	Pathway	Function	Role in IIRI
miR-21	A kind of promoting miRNA	Inhibits PTEN expression and PDCD4 expression	Activating PI3K/AKT pathway	Regulates cell proliferation, apoptosis, and inflammatory reaction	miR-21 may function by inhibiting ROS generation, alleviating cellular apoptosis, and reducing inflammatory responses
miR-29a	N/A	Inhibits the expression of COL3A1 and COL1A1	N/A	Regulates collagen expression	miR-29a may regulate the expression levels of collagen proteins, influencing the repair and reconstruction processes of the intestinal mucosa
miR-93	N/A	Inhibits the expression of PTEN and other negative regulatory genes	Promotes the activation of PI3K/AKT pathway	Participates in regulating angiogenesis, cell proliferation, and anti-apoptosis	miR-93 may contribute to the restoration of intestinal tissue function by promoting vascular regeneration and inhibiting cell apoptosis
miR-125b	N/A	N/A	Regulating NF-κB signaling pathway	Inhibits the expression of inflammatory factors (such as TNF-α and IL-6), reduces inflammation and cellular damage, and participates in regulating inflammatory responses and cell apoptosis	miR-125b may protect the intestinal mucosa from injury by inhibiting the expression of inflammatory cytokines and regulating apoptosis pathways
miR-142-3p	N/A	N/A	N/A	Plays a role in immune regulation and inflammatory responses	miR-142-3p may influence post-injury inflammation levels and tissue repair processes by regulating the activation state of inflammatory cells and immune cell responses
miR-155	Key inflammatory-associated miRNA	N/A	Regulating TLR signaling pathway and activation of NF-κB	Participates in regulating the activation of immune cells and the expression of inflammatory factors	miR-155 may influence the recovery of intestinal tissue and post-inflammatory repair processes by regulating the intensity and timing of inflammatory responses
miR-200a	N/A	N/A	Inhibits the expression of ZEB1 and ZEB2	N/A	miR-200a may promote the restoration and protection of intestinal mucosal barrier integrity and function by inhibiting the expression of ZEB1 and ZEB2, thereby regulating the epithelial-mesenchymal transition (EMT) process and cell migration capability in intestinal epithelial cells
miR-210	A kind of promoting miRNA	Affects the expression of VEGF in extracellular matrix	N/A	Participates in regulating EMT and cell migration	miR-210 may contribute to the protection and repair of injured intestinal tissue by promoting cellular hypoxia adaptation and vascular regeneration capability
miR-347	N/A	N/A	N/A	It may be involved in regulating cell metabolism and immune response	The mechanism of action of miR-347 may involve regulating cellular energy metabolism and immune cell activity, influencing the repair process of the intestine after injury

miRNA: MicroRNA; ROS: Reactive oxygen species; TLR: Toll-like receptor; VEGF: Vascular endothelial growth factor; IIRI: Intestinal ischemia-reperfusion injury.

miRNAs such as miR-93 regulate multiple genes involved in the cell cycle, enhancing reperfusion recovery in ischemic tissues and highlighting their role in promoting angiogenesis and tissue repair[67]. Conversely, upregulation of miR-347 promotes neuronal apoptosis post-ischemia, indicating its role in regulating cell death pathways and its potential as a therapeutic target for neuroprotection[68].

Significance for diagnosis and treatment

Due to their stability in body fluids and specific expression patterns in response to ischemic stress, miRNAs hold significant potential for diagnosing IIRI. For instance, changes in levels of miR-21 and miR-210 can serve as indicators of myocardial damage and predict patient prognosis[8]. Differential expression of miRNAs between reperfusion and ischemic stages underscores their diagnostic value in assessing the extent and timing of tissue damage[69].

Modulating the expression of specific miRNAs offers promising therapeutic strategies to mitigate IIRI. For example, inhibiting miR-155 can reduce inflammatory damage and improve tissue recovery during reperfusion[66]. Delivery of miRNA mimics or inhibitors via nanoparticles or viral vectors effectively alters target miRNA expression, offering a potential approach to protect tissues from ischemic injury and enhance repair processes[70].

COMPREHENSIVE INSIGHTS FROM RNA-SEQ STUDIES

Advantages and applications of RNA-seq in IIRI research

RNA-seq technology offers a comprehensive and high-throughput method for gene expression analysis[71]. It enables the unbiased identification of differentially expressed genes, including those encoding ECM proteins, as well as ncRNAs such as miRNAs, lncRNAs, and circular RNAs. With its high sensitivity, RNA-seq can detect low-abundance transcripts, making it an invaluable tool for studying complex biological processes, such as IIRI[72]. In comparison to traditional techniques like microarrays, RNA-seq provides higher resolution, the ability to discover novel transcripts, and accurate quantification of gene expression levels[73].

RNA-seq has significantly advanced IIRI research by providing detailed insights into gene expression dynamics at various stages of injury and recovery. By uncovering the underlying molecular mechanisms of tissue damage and repair, RNA-seq contributes to the identification of potential biomarkers and therapeutic targets[74]. Pivotal RNA-seq studies have revealed distinctive expression profiles of ECM genes and ncRNAs, which play essential roles in critical processes such as apoptosis, autophagy, oxidative stress, and ECM remodeling during IIRI[10-12].

Key findings in RNA-seq studies related to ECM and miRNAs

RNA-seq studies have revealed distinct expression profiles of ECM genes in IIRI[54]. For example, specific ECM gene sets are upregulated during the reperfusion phase, emphasizing their critical role in tissue repair and remodeling[55]. These studies have highlighted the dynamic nature of ECM remodeling, with gene expression changes linked to various stages of injury and recovery. Such insights are essential for the development of targeted therapies that aim to modulate ECM remodeling and improve clinical outcomes[56].

Similarly, RNA-seq has uncovered significant alterations in miRNA expression profiles during IIRI[57]. In a global miRNA expression analysis of a mouse model of hepatic IIRI, 69 differentially expressed miRNAs were identified, shedding light on the complexity of the injury response and suggesting potential strategies for targeted intervention[8].

Integrated analysis of ECM and miRNA data

Comprehensive analysis of RNA-seq data has revealed important associations between specific ECM gene sets and miRNA networks. These interactions are crucial for understanding the regulation of ECM remodeling during IIRI and for identifying key regulatory nodes that could serve as potential therapeutic targets[75]. For example, specific miRNAs can regulate ECM components, directly influencing cellular responses to injury, such as proliferation, migration, and apoptosis, thereby affecting tissue repair and regeneration processes[76,77] (Figure 1).

Figure 1 Effects exerted by some microRNAs (miR-21 and miR-93) in intestinal ischemia-reperfusion injury can inhibit PTEN and other negative regulatory gene expression, promoting activation of the PI3K/AKT pathway. This facilitates vascular regeneration and suppresses apoptosis, contributing to the restoration of intestinal tissue function. miR-21 potentially inhibits reactive oxygen specie generation, alleviating cell apoptosis and inflammatory responses. miR-29a suppresses COL3A1 and COL1A1 expression to regulate matrix metalloproteinase expression, thereby modulating collagen content in the extracellular matrix. By adjusting collagen expression levels, it affects the repair and reconstruction processes of the intestinal mucosa. miR-200a participates in regulating epithelial-mesenchymal transition and cell migration by inhibiting ZEB1 and ZEB2 expression, thus promoting the restoration and protection of the intestinal mucosal barrier, which helps maintain its integrity and functionality. ECM: Extracellular matrix.

Systems biology approaches, including network analysis and computational modeling, integrate RNA-seq data with other omics datasets, providing a comprehensive view of molecular interactions and regulatory networks in IIRI[10]. This integrated analysis has clarified the complex interplay between ECM remodeling and miRNA regulation, offering insights into how these processes contribute to tissue damage and recovery[78].

Case studies and examples

Several RNA-seq studies have been conducted in experimental settings to explore innovative therapeutic strategies. For example, a study examining the miRNA expression profile following hepatic IIRI in mice revealed the temporal dynamics of miRNA regulation during reperfusion. This research identified miR-29a and let-7 as key regulators of apoptosis through their interaction with IGF-1, underscoring the therapeutic potential of miRNA-based approaches[79]. Another study on the role of lncRNAs in myocardial IIRI highlighted their critical involvement in modulating inflammatory responses and cell survival pathways, suggesting that targeting lncRNAs could offer promising therapeutic strategies to mitigate IIRI[80].

Moreover, ECM-related biomarkers identified through RNA-seq have facilitated the development of diagnostic tools for the early detection of IIRI, enabling timely and targeted interventions to improve patient outcomes[78]. In preclinical models of IIRI, the modulation of specific miRNA expression using mimics or inhibitors has shown promise for translating these findings into clinical therapies[66].

FUTURE PERSPECTIVES AND RESEARCH DIRECTIONS

Advances in RNA-seq technology

Recent advancements in RNA-seq technology, including single-cell RNA-seq (scRNA-seq) and spatial transcriptomics, have significantly enhanced our capability to analyze gene expression with unparalleled resolution and context. These technologies can discern cell-specific responses during IIRI and map the spatial distribution of gene expression changes within tissue structures[81]. Integrating RNA-seq with other omics technologies such as proteomics and metabolomics holds promise for providing a more comprehensive understanding of the underlying molecular mechanisms of IIRI. This multi-omics approach can reveal new therapeutic targets and biomarkers for early diagnosis, enabling personalized treatments[10].

Translational research

Translational research aims to bridge the gap between fundamental scientific discoveries and clinical applications. In the context of IIRI, this includes validating RNA-seq findings in preclinical models and developing clinical trials to test the efficacy of potential therapies[80]. Developments in RNA interference therapy, targeting specific gene expression involved in IIRI, illustrate a path from laboratory research to clinical applications. These therapies have shown promise in preclinical studies to reduce inflammation and tissue damage in multiple organs including the heart, liver, and kidneys[82].

Clinical trials are crucial for evaluating the safety and efficacy of new therapies based on RNA-seq discoveries. For example, ongoing trials investigate the use of miRNA mimics or inhibitors to modulate critical miRNA expression involved in IIRI, potentially offering new therapeutic options for patients[83]. Experimental therapies targeting ECM remodeling and miRNA regulatory networks show promising results in preclinical models, highlighting their potential in improving outcomes of IIRI. Continued research and clinical validation are essential for translating these therapies into standard clinical practices[81].

Personalized medicine

Personalized medicine aims to customize diagnostic and treatment strategies based on individual genetic and molecular profiles. Integrating RNA-seq data with specific clinical information can facilitate personalized treatment plans for IIRI, enhancing efficacy and minimizing side effects[78]. Biomarkers identified through RNA-seq studies, such as specific miRNAs and ECM components, can serve as diagnostic tools to predict individual responses to treatments and monitor disease progression. This personalized approach enables clinicians to make informed decisions and optimize treatment strategies for each patient[79].

Customizing treatments based on individual genetic and molecular characteristics involves identifying unique gene expression patterns related to IIRI using RNA-seq data. This information can guide the selection of targeted therapies aimed at specific molecular mechanisms for each patient[84]. Advances in gene editing technologies like CRISPR-Cas9 provide additional opportunities to develop personalized therapies by precisely modifying disease-related genes. Combining these technologies with insights from RNA-seq enhances our ability to correct genetic abnormalities and improve patient outcomes[81].

Limitations

While this review systematically summarizes the diagnostic and therapeutic potentials of ECM gene sets and miRNA regulatory networks in IIRI, it has several limitations. It relies on published studies, potentially overlooking the latest or ongoing research. Various experimental models of IIRI vary, which may affect the feasibility of translating research results into clinical practice. Additionally, heterogeneity in data across studies, including experimental conditions and analysis methods, may impact the coherence and reliability of review findings. Despite demonstrating diagnostic and therapeutic potentials in preclinical studies, ECM genes and miRNAs require further validation for effectiveness and safety in clinical applications. Clinical trial data are still relatively lacking, potentially facing unknown challenges in practical applications. The review acknowledges the possibility of selective bias despite efforts to cover multiple viewpoints and research outcomes. Moreover, the roles of ECM and miRNAs in IIRI involve complex regulatory networks, posing challenges for comprehensively covering all related pathways and mechanisms in this review. Addressing these limitations in future research is essential to deepen our understanding of IIRI pathophysiology and advance the development of relevant diagnostic and therapeutic technologies.

CONCLUSION

As a major challenge in clinical surgery, the complex pathophysiological mechanisms and high-risk clinical outcomes of IIRI necessitate continuous exploration of new diagnostic and treatment strategies. This review explores the latest advancements in ECM gene sets and miRNA regulatory networks in IIRI, aiming to reveal their molecular impacts and clinical applications. Leveraging frontier developments in RNA-seq technology, we elucidate the significant value of ECM and miRNA in diagnosing and treating IIRI.

ECM not only serves as the structural basis of tissues but also plays a dual role in regulating inflammation, cell apoptosis, and tissue repair. Specific miRNAs such as miR-125b and miR-200a highlight promising targets for intervention in IIRI. Furthermore, integration of RNA-seq and other omics technologies enables a finer resolution of dynamic ECM gene and miRNA expression in IIRI, facilitating the discovery of potential biomarkers for early diagnosis and prognosis assessment.

Future research directions should prioritize using scRNA-seq and spatial transcriptomics to map molecular landscapes in IIRI, focusing on deciphering cell-specific and tissue microenvironmental influences on disease progression. Additionally, translational research should be strengthened by translating RNA-seq-based discoveries into clinical applications and validating the efficacy and safety of ECM modulation drugs and miRNA-targeted therapies through carefully designed clinical trials. Lastly, integrating patient-specific clinical information with multi-omics data can tailor more precise treatment strategies, optimizing outcomes and minimizing side effects.

In summary, ECM gene sets and miRNA regulatory networks offer new hope for diagnosing and treating IIRI, yet fully harnessing their potential requires ongoing scientific advancement and continuous validation in clinical practice. Future research should address current limitations, deepen our understanding of IIRI pathophysiology, and advance related diagnostic and therapeutic technologies.