Early views held that mature miRNAs localize exclusively in the cytoplasm. However, in 2004, Meister et al. reported that ~20% of miR-21 resides in the nucleus of HeLa cells, challenging this classic notion^[4]. Subsequent studies have further confirmed that various mature miRNAs can localize to the nucleus and exert distinct biological functions. For example, nucleolar miR-206 participates in rRNA processing, and high-throughput sequencing revealed consistent nuclear and cytoplasmic miRNA profiles^[5,24,25]. Moreover, the existence of functional nuclear RISC has been validated, with nuclear let-7 cleaving a fully complementary target at 86% efficiency vs. 60% in the cytoplasm^[26]. Collectively, these findings show that nuclear miRNAs are widespread and biologically active.

Research on the nuclear import mechanisms of miRNAs remains limited, which can be categorized into two types: sequence motif-dependent pathways and protein-mediated pathways. In the sequence motif-dependent pathway, the 3' terminal AGUGUU motif of miR-29b alone facilitates its nuclear localization^[27]. Transferring this motif to the normally cytoplasmic miR-29a or to a small interfering RNA enriches them in the nucleus^[27]. Another study identified a conserved 3' ASUS motif (where S denotes C or G) in nuclear-enriched miRNAs from neural precursor cells^[28]. Regarding protein-mediated pathways, NRDE-3 in C. elegans can guide small RNAs into the nucleus^[29]. In mammals, importin 8 selectively mediates the nuclear import of Argonaute 2 (Ago2) complexes loaded with specific miRNAs such as miR-709 and miR-29b^[30,31]. TNRC6A (a paralog of GW182) functions as a critical nucleocytoplasmic shuttling adaptor that directly modulates Ago2 subcellular localization and mediates the nuclear import of miRNAs^[26,27]. In the cytoplasm, the association of Ago2 with TNRC6A mutually inhibits their nuclear import to maintain a regulatory balance, which is also influenced by cell density^[32,35]. Moreover, the nuclear miRNA-Ago2 complex, upon binding to nascent target transcripts, prolongs its retention in the nucleus^[36]. Collectively, diverse pathways mediate the nuclear import of miRNAs. However, a unified mechanism remains poorly understood, and how these pathways coordinate is unclear, highlighting a need for further research.

Nuclear miRNAs have been confirmed to regulate gene expression at the transcription level either positively or negatively. They are increasingly recognized as key regulators of nuclear RNA metabolism, participating in autoregulatory feedback loops, mediating post-transcriptional silencing of nuclear-retained transcripts, and influencing pre-mRNA splicing decisions.

Atherosclerosis constitutes a chronic inflammatory disorder of the arterial wall driven by multiple risk factors, including hyperlipidemia, hypertension, smoking, diabetes, and genetic susceptibility^[56,57]. Lesions often localize to regions of disturbed blood flow (such as arterial bifurcations), where turbulent shear stress can induce endothelial dysfunction^[58]. Endothelial cell proliferation and regenerative capacity are essential for maintaining vascular integrity and preventing lesion formation. Among miRNAs enriched in endothelial cells, miR-126 plays a central role in angiogenesis and vascular homeostasis^[59-61]. Derived from pre-miR-126, miR-126-5p plays a well-established role in angiogenesis and has been shown to exert atheroprotective effects through spatially distinct mechanisms^[62]. The expression of miR-126-5p is modulated by hemodynamics^[62]. Researchers divided the aorta of high-fat diet-fed Apoe^-/- mice into atherosclerosis-resistant regions (laminar flow regions, straight segments of the thoracic and abdominal aorta) and atherosclerosis-susceptible regions (turbulent flow regions, branch points of the aortic arch, aortic root, and carotid arteries)^[62]. Quantitative real-time PCR (qRT-PCR) results revealed that the laminar flow-dominated resistant regions exhibited high expression of miR-126-5p, accompanied by low mRNA and protein levels of Dlk1, whereas the opposite pattern was observed in turbulent flow-prone susceptible regions^[62]. No significant difference in miR-126-3p expression was detected between the two types of regions, indicating that blood flow specifically regulates miR-126-5p alone^[62]. Functionally, miR-126-5p targets Dlk1 mRNA via the RISC in the cytoplasm, relieving the inhibition of Notch1 and promoting endothelial cell regeneration^[62]. Beyond this canonical cytoplasmic role, miR-126-5p can also be imported into the nucleus under high shear stress (HSS) or rapamycin-induced autophagy via an Ago2:Mex3a-containing complex, as validated in HUVECs^[47]. In vitro, nuclear miR-126-5p dissociates to target monomeric caspase-3, inhibiting its activity and reducing endothelial apoptosis^[47]. Consistently, in ATG5EC-KO mice and Mex3a^-/- mice, the nuclear enrichment ability of miR-126-5p is significantly reduced, leading to exacerbated endothelial apoptosis and aggravated atherosclerotic lesions^[47]. Notably, in human carotid artery samples, miR-126-5p and caspase-3 exhibit markedly stronger co-localization signals in non-lesioned regions than in atherosclerotic regions^[47]. Collectively, these findings establish that miR-126-5p orchestrates endothelial protection against atherosclerosis through a spatially coordinated, dual-compartment strategy.

Foam cell formation derived from macrophages is a central pathological feature of atherosclerosis^[63,64]. Scavenger receptors (SR-A and CD36) mediate the cellular uptake of oxidized low-density lipoprotein (oxLDL), a critical step in foam cell development^[56,65]. As a member of the nuclear factor of activated T-cells (NFAT) protein family, NFATc3 functions as a transcription factor triggered by the Ca²⁺/calcineurin signaling pathway in T cells^[66-68]. Liu et al. (2021) found that the transcription factor NFATc3 in macrophages acts as a negative regulator of atherosclerosis^[37]. The expression of NFATc3 is markedly reduced in human carotid plaques and in aortic lesions of hyperlipidemic Apoe^-/- mice, and lower NFATc3 mRNA levels in peripheral blood mononuclear cells correlate significantly with symptomatic plaque progression^[37]. Consistent with a protective role, after high-fat diet treatment, NFATc3MKO mice display larger atherosclerotic plaques, increased necrotic cores, and reduced plaque stability compared with control mice of the same genotype^[37]. In contrast, NFATc3MTG mice presented markedly alleviated aortic lesions and suppressed local vascular inflammation upon high-fat diet challenge^[37]. Meanwhile, the proportions of SR-A- and CD36-positive macrophages within plaques and foam cell formation are increased in NFATc3MKO mice, whereas the opposite phenotype is observed in NFATc3MTG mice^[37]. Mechanistically, NFATc3 deficiency upregulates the scavenger receptors SR-A and CD36, promoting oxLDL uptake and foam cell formation^[37]. NFATc3 exerts its atheroprotective effect by directly binding to the promoter of miR-204 and driving its transcription^[37]. This activation initiates a spatially organized regulatory circuit: In the cytoplasm, the miR-204-5p strand post-transcriptionally silences SR-A via canonical 3' UTR targeting^[37]. Simultaneously, the miR-204-3p strand translocates to the nucleus, where it binds to the CD36 promoter, displaces Ago2, and reduces active histone marks (H3K27ac and H3K4me3), ultimately repressing CD36 transcription^[37]. Adeno-associated viruses (AAV)-mediated pre-miR-204 treatment for 8 weeks effectively reversed the upregulated expression of SR-A and CD36 in peritoneal macrophages of high-fat diet-fed NFATc3MKO mice^[37]. This intervention also reduced aortic atherosclerotic plaque area and enhanced plaque stability in these mice^[37]. In contrast, inhibition of miR-204 in NFATc3MTG mice abolished the protective effects of NFATc3^[37]. Consequently, the NFATc3/miR-204 axis represents a coordinated defense mechanism against foam cell formation, simultaneously engaging post-transcriptional and chromatin-based silencing to downregulate two critical scavenger receptors, revealing a multifaceted transcriptional epigenetic barrier against atherosclerotic progression.

In addition to anti-atherogenic function, nuclear miRNAs can also exert pro-atherogenic effects, as exemplified by a pathogenic cascade initiated by the periodontal bacterium Porphyromonas gingivalis (P. gingivalis)^[48,69-71]. In clinical samples, P. gingivalis can be found in more than 80% of atherosclerotic plaques^[70,72,73]. Apoe^-/- mice infected with P. gingivalis exhibited increased aortic plaque area, accompanied by elevated levels of miR-143/145 and increased apoptotic cells within the lesions^[48]. In vitro, vascular smooth muscle cells (SMCs) are more susceptible to P. gingivalis-induced apoptosis than endothelial cells^[48]. P. gingivalis activates the TLR2-NF-κB signaling cascade to promote SMC apoptosis in a caspase-dependent manner, and mediates the extracellular release of nuclear miR-143/145 via apoptotic extracellular vesicles^[48]. Macrophages internalize these vesicles, after which miR-143/145 translocate to the nucleus and assemble into a functional complex with Ago2^[48]. This nuclear miRNA-Ago2 complex binds specific promoter sites of the Siglec-G gene, recruits Pol II, and upregulates Siglec-G transcription^[48]. The resulting elevation of Siglec-G on macrophages inhibits their efferocytic capacity, leading to impaired clearance of apoptotic SMCs^[48]. In miR-143/145^-/-Apoe^-/- double-knockout mice, the number of Siglec-G-positive macrophages is decreased, the efferocytosis capacity of macrophages is restored, and aortic atherosclerotic lesions are alleviated^[48]. Conversely, overexpression of miR-143/145 exacerbated atherosclerotic lesions in mice^[48]. This pathway highlights a novel intercellular communication mechanism in which nuclear miRNAs, transferred via apoptotic vesicles, act as transcriptional co-regulators in recipient macrophages to disrupt tissue homeostasis and promote inflammatory disease progression.

The lncRNA atherosclerotic plaque pathogenesis-associated transcript (APPAT) is detectable in circulation, and its levels correlate with atherosclerotic progression^[74,75]. Functionally, APPAT overexpression suppresses the oxLDL-induced migratory and proliferative capacity of HASMCs, whereas overexpression of miR-647 promotes these pro-atherogenic phenotypes^[49]. Bioinformatic analysis and dual-luciferase reporter assays validate the direct reciprocal suppression between APPAT and miR-647^[49]. Dual-luciferase reporter assays confirm that miR-647 binds to the 3'-UTR of FGF5 and upregulates its mRNA and protein levels, while APPAT overexpression reverses ox-LDL-induced elevation of FGF5^[49]. Notably, subcellular fractionation shows that miR-647 localizes to both the nucleus and cytoplasm of HASMCs, with a relatively lower abundance in the cytoplasmic compartment^[49]. This distribution intriguingly suggests that the positive regulation of FGF5 by miR-647 may occur via a non-canonical nuclear pathway, potentially involving direct gene activation^[49]. However, given its detectable cytoplasmic presence, a complementary canonical mechanism, in which miR-647 indirectly elevates FGF5 by repressing its suppressors via the RISC pathway, cannot be excluded^[49]. Thus, the APPAT/miR-647/FGF5 axis orchestrates a multi-tiered regulatory circuit that fine-tunes smooth muscle cell phenotypes in atherosclerosis by integrating a lncRNA-mediated checkpoint with the dual-compartment actions of a miRNA, underscoring the sophisticated layering of RNA-based control in vascular disease.

Myocardial remodeling, a key pathophysiological process in heart failure, involves extensive transcriptomic reprogramming, most notably the pathological switch from the adult α-myosin heavy chain (MYH6) to the fetal β-myosin heavy chain (MYH7)^[76-78]. Existing research demonstrates that pressure overload significantly upregulates nucleus-localized Ago2 in cardiomyocytes, as observed in human dilated cardiomyopathy patients and transverse aortic constriction (TAC) mouse models^[38]. Within the nucleus, Ago2 functions as a transcriptional co-activator. It is recruited to the promoter region of ANKRD1 through an interaction mediated by nuclear miR-92a-3p^[38]. This miRNA guides Ago2 by binding at the ANKRD1 locus, thereby enhancing ANKRD1 transcription^[38]. The interactions between miR-92a-3p, Ago2, and ANKRD1 are observed in HL-1 cells^[38]. The upregulated ANKRD1 protein, in turn, functions as a transcriptional co-activator that binds specifically to the MYH7 promoter, selectively increasing MHC7 expression without affecting MHC6^[38]. The consequent rise in the MHC7/MHC6 ratio impairs myocardial contractility, promotes hypertrophy and fibrosis, and accelerates the transition from adaptive cardiac hypertrophy to decompensated heart failure^[38]. A recombinant adeno-associated virus serotype 9 (rAAV9)-mediated knockdown of Ago2 or ANKRD1 effectively reverses the heart failure phenotype induced by TAC^[38]. Intervention with ivermectin (which inhibits the nuclear import of Ago2 and ANKRD1) or synthetic ANKPep (a peptide mimicking the nuclear localization signal of ANKRD1 that specifically blocks ANKRD1 nuclear translocation) in TAC mice both ameliorated cardiac function and myocardial hypertrophy^[38].

Correspondingly, nuclear primary miRNAs also participate in transcriptional reprogramming during cardiac hypertrophy. A prominent example is pri-miR-208b, which modulates pathological cardiac remodeling through direct chromatin interactions^[50]. During TAC-induced cardiac hypertrophy, Ezh2, a Polycomb-group protein, binds to the bidirectional promoter (bdP) of the α-myosin Heavy Chain (α-MHC) and β-MHC (antisense), facilitating H3K27me3 to suppress α-MHC and β-MHC expression^[50]. pri-miR-208b reinforces this repression by interacting with both Ezh2 and the bdP, as in vitro experiments confirm direct binding of pri-miR-208b to the -1227 to -1330 region of bdP, and this interaction can be disrupted by RNase H treatment^[50]. The histone deacetylase inhibitor Trichostatin A (TSA) disrupts the interaction between Ezh2 and bdP, which diminishes chromatin occupancy of pri-miR-208b. Consequently, TSA treatment reverses the aberrant gene expression associated with pathological cardiac hypertrophy in TAC mice^[50]. Notably, antisense β-MHC (AS β-MHC) can associate with chromatin independently of Ezh2 and pri-miR-208b, and its knockdown does not significantly alter Ezh2-promoter interaction^[50]. Interestingly, a recent study reveals a sex-specific regulatory mechanism in cardiac tissue. Female mouse left ventricles exhibit substantially higher nuclear pri-miR-208b abundance relative to male counterparts^[79]. This sex difference is independent of DNA methylation but relies on sex-specific methylation of the lncRNA Mhrt^[79]. In female mice, methylation of the CG18 locus within Mhrt facilitates its binding to methyl-CpG-binding protein MeCP2^[79]. Through an RNA-dependent mechanism, the MeCP2 complex subsequently recruits corepressors (Rest and HDAC2) to the pri-miR-208b promoter, ultimately alleviating transcriptional repression^[79]. Given the established role of pri-miR-208b in regulating the pathological Myh6/Myh7 switch, these observations suggest that nuclear miRNA-guided epigenetic mechanisms may contribute to sex-based differences in cardiovascular disease susceptibility^[50,79]. Nevertheless, this study exhibits distinct limitations. In particular, the hormonal regulation of Mhrt CG18 methylation remains undefined, and its stability in the context of hypertrophy and other pathological conditions has yet to be validated.

Phosphatase and tensin homolog (PTEN) is widely recognized as a tumor suppressor^[80,81]. It is also abundantly expressed in various cardiovascular cell types, including cardiomyocytes, fibroblasts, vascular smooth muscle cells, and endothelial cells, and functions as a potent negative regulator of cardiac hypertrophy^[82,83]. PTEN maintains cardiomyocyte homeostasis and modulates myocardial energy metabolism via inhibiting the PI3K/Akt/mTOR signaling axis^[82-84]. While cytoplasmic miRNAs such as miR-217 are known to post-transcriptionally suppress PTEN to facilitate cardiac hypertrophy, a recent study reveals that nuclear miRNAs can also target PTEN through a distinct regulatory mechanism^[51,84]. miR-665 represents a typical example of such nuclear regulatory miRNAs. In TAC mice, nuclear-enriched miR-665 is markedly upregulated and drives pathological cardiac remodeling by directly repressing PTEN in cardiomyocytes^[51]. Elevated miR-665 exacerbates cardiomyocyte hypertrophy, promotes myocardial fibrosis, and aggravates cardiac dysfunction, as validated in both cellular models and TAC-induced mouse models^[51]. Mechanistically, miR-665 represses PTEN transcription independent of the canonical 3' UTR pathway^[51]. Instead, it binds two evolutionarily conserved regions within the PTEN promoter: site 1 (-217/-211) and site 2 (-835/-827), and recruits Ago2 to inhibit PTEN expression at the transcriptional level. These regulatory effects are validated by dual-luciferase reporter assays and ChIP assays^[51]. Restoration of PTEN expression reverses miR-665-induced cardiac dysfunction, cardiomyocyte hypertrophy and myocardial fibrosis in TAC mice, highlighting the pivotal role of this axis in pathological cardiac progression^[51].

Diabetic cardiomyopathy represents a common form of secondary cardiomyopathy, characterized by the synergistic pathophysiological effects of hyperglycemia-induced glucotoxicity and dyslipidemia-associated lipotoxicity^[85]. These pathological processes collectively induce myocardial structural and functional abnormalities of the myocardium via oxidative stress and mitochondrial dysfunction^[85]. CD36, a critical transmembrane transporter for long-chain fatty acids (FAs), facilitates free fatty acids (FFAs) uptake in cardiomyocytes and provides substrates for fatty acid oxidation^[86,87]. Cardiomyocyte FFA uptake is markedly reduced in MHC-PPARα/CD36^-/- mice^[86]. Consistently, CD36 mRNA and protein levels are elevated in insulin-resistant primary cardiomyocytes^[86]. shRNA-mediated CD36 knockout decreases cellular uptake of long-chain free fatty acids by approximately 50% and greatly alleviates intracellular lipid accumulation, while silencing other fatty acid transporters fails to produce comparable improvements in lipotoxicity^[86]. A key factor driving this pathological CD36 upregulation is nuclear-enriched miR-320, which acts as a small activating RNA (saRNA) that directly binds to the CD36 promoter and enhances its transcription^[52]. This effect promotes fatty acid uptake and exacerbates lipotoxicity, thereby accelerating diabetic cardiomyopathy progression. Therapeutic intervention with a rAAV9-delivered miR-320 inhibitor (TuD) effectively restores cardiac function in db/db mice^[52]. Interestingly, a positive feedback loop exists between CD36 and miR-320^[53]. Elevated CD36 promotes reactive oxygen species (ROS) generation, and further activates the transcription factor SP1^[53]. SP1 in turn binds to the miR-320 promoter, further increasing miR-320 expression and thereby reinforcing CD36 transcription^[53]. This CD36-ROS-SP1-miR-320 axis forms a self-sustaining cycle that exacerbates lipotoxicity^[53]. CD36 serves as the initiator of this feedback loop. In STZ-induced diabetic mice, CD36 protein levels are significantly increased within 2 weeks of hyperglycemia onset, while miR-320 upregulation is delayed until four weeks^[53]. Correspondingly, high-glucose-treated AC16 cardiomyocytes exhibit CD36 protein accumulation prior to elevated CD36 mRNA levels^[53]. This early CD36 protein elevation is closely associated with high glucose-induced extracellular Ago2 secretion, which reduces intracellular Ago2 availability^[53]. Consequently, diminished Ago2 binding to CD36 mRNA alleviates translational repression, thereby enhancing CD36 protein synthesis^[53].

Chronic orthostatic intolerance syndromes, including the classic subtype postural orthostatic tachycardia syndrome (POTS), are characterized by persistent dizziness, palpitations, and fatigue upon prolonged standing, which arises from cardiac sympathetic dysautonomia^[88,89]. A hallmark of POTS is impaired synaptic clearance of norepinephrine (NE), leading to excessive sympathetic tone in the upright position^[88]. Genetic investigations have identified specific molecular alterations responsible for disrupted NE homeostasis^[90]. A genetic analysis of a family with orthostatic intolerance identified a G237C missense mutation (Ala457Pro) in exon 9 of the norepinephrine transporter (NET) gene (SLC6A2)^[90]. This mutation abolishes over 98% of NET uptake activity, resulting in severely reduced NE clearance, elevated upright plasma NE levels, and an altered DHPG/NE ratio, directly linking defective NET function to familial orthostatic tachycardia and NE dysregulation^[90]. Beyond genetic defects, epigenetic mechanisms further contribute to NET deficiency in POTS. In patients’ leukocytes, the nuclear microRNA let-7i forms a complex with MeCP2 that binds to the NET promoter, where it facilitates histone deacetylation and represses NET transcription^[54]. Although this regulatory axis has been corroborated in HeLa cells, its direct role in modulating NET expression in patient-specific sympathetic tissues awaits further validation^[54]. Consistent with these findings, immunohistochemical analyses of peripheral sympathetic nerves from POTS patients reveal reduced NET protein expression and blunted sympathetic responsiveness to postural challenge, even without elevated systemic NE spillover^[91]. This dissociation between nerve activity and NE release indicates that NET dysfunction, whether inherited or epigenetically acquired, is a core pathophysiological disturbance that disrupts sympathetic homeostasis and triggers the clinical presentation of orthostatic intolerance.

DNA methyltransferase Dnmt3b is essential for mammalian embryogenesis^[92]. Loss of Dnmt3b leads to Immunodeficiency, Centromeric instability, and Facial anomalies syndrome (ICF syndrome)^[93]. Dnmt3b-knockout mice exhibit embryonic lethality at E14.5-E16.5 with typical ventricular septal defects and multi-organ hypoplasia, highlighting its non-redundant role in cardiac morphogenesis^[93]. In HL-1 cells, inhibition of canonical Wnt signaling (e.g., via IWR-1) triggers IPO8/Ago2-mediated nuclear translocation of miR-133a^[55]. Nuclear miR-133a binds to a specific promoter-associated sequence (PBSM) of Dnmt3b and epigenetically represses its expression through H3K27 trimethylation and disruption of a Dnmt3b autoregulatory feedback loop^[55]. This regulatory pathway has been validated in human embryonic stem cell-derived cardiomyocytes, where nuclear miR-133a-mediated Dnmt3b suppression alters the expression of key maturation genes, including contractile components (Myh6, Tnni2) and calcium-handling elements (Cacna1c), and affects the subcellular localization of actin and calcium channels, thereby modulating cardiomyocyte structural and functional maturation^[55]. Taken together, these results define a pathogenic cascade whereby Wnt signaling suppression triggers nuclear miR-133a-mediated epigenetic silencing of Dnmt3b, resulting in defective cardiomyocyte gene expression^[55]. Since Dnmt3b is indispensable for ventricular septation, dysregulation of this axis thus identifies miR-133a-mediated transcriptional control as a potential etiological contributor to congenital cardiac malformations.

In conclusion, nuclear miRNAs play key regulatory roles across a spectrum of cardiovascular diseases, including atherosclerosis, heart failure, diabetic cardiomyopathy, POTS, and so forth. A common feature is that most nuclear miRNAs assemble sequence-specific guiding complexes with Ago2 as the core scaffold protein. Through direct binding to gene promoters or physical interactions with chromatin-associated factors, such miRNAs engage chromatin and modulate the transcription of target genes in either an activating or repressive manner. Despite sharing similar molecular mechanisms, nuclear miRNAs exhibit strong context-dependent regulatory effects. The regulatory effects of these nuclear miRNAs on target genes, molecular functions, and downstream biological consequences are fully determined by disease categories, cellular phenotypes, and upstream signaling cascades [Table 1]. Such discrepancies arise from divergent co-regulatory molecules recruited to miRNA-loaded complexes. Even the identical target gene may exhibit opposing regulatory outputs under different pathological conditions. CD36 serves as a typical example. In atherosclerosis, nuclear-localized miR-204-3p binds to the CD36 promoter and suppresses its transcription, thereby reducing macrophage foam cell formation and slowing plaque progression^[37]. In contrast, miR-320 functions as a saRNA to upregulate CD36 expression in diabetic cardiomyopathy, exacerbating myocardial lipotoxic injury^[52]. These findings firmly prove that nuclear miRNAs exhibit disease-specific regulatory patterns matching the corresponding pathophysiological traits.

Instead of modulating individual molecular targets in isolation, nuclear miRNAs achieve precise transcriptional control via multi-layered, multi-compartmental coordinated regulatory networks, which can be categorized into three canonical regulatory modes. The first pattern refers to dual compartmental functions of a single miRNA molecule. miR-126-5p exerts distinct activities in the cytoplasm and nucleus. In the cytoplasm, it targets and represses Dlk1 via RISC to maintain endothelial proliferation and mitigate plaque formation^[62]. In the nucleus, it directly binds and inhibits caspase-3, thereby suppressing endothelial apoptosis^[47]. As such, one miRNA molecule concurrently modulates two signaling cascades, markedly improving regulatory efficiency. The second pattern lies in functional differentiation between two strands of one miRNA. For example, the two strands of miR-204 establish a complementary regulatory system. Cytoplasmic miR-204-5p silences SR-A through canonical post-transcriptional pathways, while nuclear miR-204-3p represses CD36 at the transcriptional level, jointly restraining foam cell generation^[37]. Such strand-specific functional divergence is also observed in the miR-126 family. Cytoplasmic miR-126-3p binds Ago2 to target SPRED1 and PIK3R2, relieving the inhibition of the VEGF pathway to facilitate vascular repair and alleviate endothelial inflammation, which complements the anti-apoptotic function of nuclear miR-126-5p^[47,60]. These findings imply that strand-specific functional differentiation may represent a widespread regulatory strategy among miRNA families. The third pattern involves pathological positive feedback loops that amplify tissue damage. The CD36-ROS-SP1-miR-320 positive-feedback loop in diabetic cardiomyopathy serves as a representative example^[53]. Collectively, these spatiotemporally coordinated pathways render cardiovascular transcriptional networks highly robust. Single molecules concurrently operate at multiple regulatory nodes and hierarchical layers to enable efficient pathological signal transduction.

On this basis, nuclear miRNAs interact with lncRNAs and transcription factors to form intricate regulatory networks, such as the APPAT/miR-647/FGF5 axis and the CD36-ROS-SP1-miR-320 feedback loop, which collectively mediate pathological phenotypic switching of vascular smooth muscle cells and cardiomyocytes^[49,53]. Notably, functional diversification arises from disease-specific targeting and regulatory orientation. In atherosclerosis, nuclear miRNAs display bidirectional effects. miR-126-5p and miR-204 attenuate endothelial injury and foam cell formation, whereas miR-143/145 accelerate plaque progression via intercellular signaling, with targets centered on vascular-cell genes like Dlk1, CD36, and Siglec-G^[37,47,48]. In heart failure, nuclear miRNAs predominantly drive maladaptive remodeling by regulating myosin isoform switching (e.g., miR-92a-3p-mediated transcription activation of ANKRD1 induces MYH7 upregulation) and modulating hypertrophic signaling pathways (e.g., miR-665-mediated PTEN repression)^[38,51]. In diabetic cardiomyopathy, nuclear miRNAs primarily disrupt lipid metabolic dysregulation, as exemplified by miR-320-mediated CD36 activation that exacerbates myocardial lipotoxic injury^[52]. In POTS, nuclear let-7i uniquely targets NET, disrupting sympathetic homeostasis^[54]. An additional layer of regulatory complexity arises from sex-specific regulation. In heart failure, female-specific methylation of the lncRNA Mhrt modulates the expression of pri-miR-208b, representing a unique regulatory trait that has not been widely observed in other diseases^[79]. Thus, despite the conserved transcriptional mechanism of nuclear miRNAs, their functional outputs are precisely adapted to the cellular and molecular landscape of each cardiovascular pathology.

Overall, nuclear miRNAs function via conserved chromatin-based transcriptional regulatory mechanisms. Through cytoplasmic-nuclear compartmentalized division of labor, strand-specific functional differentiation, pathological positive feedback loops, and crosstalk with lncRNAs and transcription factors, they form multi-layered coordinated regulatory networks. Their biological functions shift dynamically in response to disease microenvironments to accommodate the pathological progression of diverse cardiovascular disorders, providing multi-dimensional theoretical foundations for developing precise cardiovascular therapeutic strategies targeting nuclear miRNAs.

miRNA-targeted therapy represents a pivotal frontier in the development of non-coding RNA-based therapeutics, demonstrating unique and irreplaceable advantages in disease treatment and offering innovative strategies for the clinical intervention of complex disorders. As endogenous regulatory molecules, miRNAs can directly harness intrinsic biogenesis and functional mechanisms without the need for exogenous regulatory systems, thereby minimizing off-target risks. Furthermore, chemical modifications such as 2'-O-methylation and LNA significantly reduce the immunogenicity of miRNAs, making them safer than certain exogenous synthetic drugs^[13,94]. A distinctive strength of miRNA-based therapeutics lies in their capacity to simultaneously regulate multiple key genes within a shared pathological pathway via a single seed sequence. This multi-targeting modality enables miRNA-based therapies to overcome the limitations of traditional single-target approaches, making them particularly suitable for treating polygenic disorders. The inherent flexibility of this approach further permits the therapeutic restoration of protective miRNAs via mimics, or the suppression of pathogenic miRNAs using anti-miRNA oligonucleotides (AMOs). When integrated with advanced delivery platforms, such as AAV or GalNAc conjugates, this strategy facilitates precise targeting of disease-driving pathways, aligning with the principles of precision medicine^[95,96]. Consequently, miRNA-targeted therapy has rapidly evolved into a promising frontier for the diagnosis and treatment of cardiovascular diseases^[95,97,98]. Several cardiovascular-relevant miRNAs have shown encouraging efficacy in preclinical models, and a number of candidates (e.g., CDR132L, Remlarsen) have progressed to Phase II clinical trials^[99,100], underscoring their substantial translational potential^[13,97].

Among the nuclear miRNAs previously discussed in relation to cardiovascular disease mechanisms, miR-92a-3p and miR-126-3p exemplify successful transitions from mechanistic insight to clinical exploration. In large-animal (porcine) ischemia-reperfusion models, anti-miR-92a-3p agents, such as LNA-modified inhibitors, significantly reduce myocardial infarction size while attenuating cardiomyocyte apoptosis and inflammation^[101]. Mechanistically, these protective effects are attributed to the repression of pro-survival genes, including BCL-2, and activation of the PI3K/Akt signaling pathway^[101,102]. The anti-miR-92a-3p candidate MRG-110 has advanced into clinical trials, where it is being evaluated for safety and efficacy in promoting angiogenesis and accelerating tissue repair in wound healing^[13]. Preclinical and early-phase clinical data indicate that subcutaneous delivery achieves target tissue distribution, robustly suppresses miR-92a-3p activity, and demonstrates a favorable safety profile, with no significant immune-related adverse events reported^[103]. miR-126-3p, predominantly expressed in vascular endothelial cells, promotes angiogenesis and maintains endothelial integrity by inhibiting SPRED1 and PI3KR2, thereby activating the VEGF pathway^[104]. Its downregulation in conditions such as myocardial infarction and atherosclerosis leads to impaired vascular repair^[104]. Notably, patients with advanced heart failure complicated by pulmonary hypertension (PH) display markedly diminished circulating miR-126-3p concentrations, a finding that correlates with higher pulmonary artery pressure and worse right ventricular function^[104]. This suggests a potential role for miR-126-3p in mitigating pulmonary hypertension-associated vascular remodeling, highlighting it as a novel therapeutic target for heart failure complications. Preclinical research is currently exploring the targeted delivery of miR-126 mimics via exosomes or aptamers (e.g., transferrin receptor aptamers) to promote angiogenesis in ischemic myocardium, demonstrating potential in improving vascular remodeling in PH^[104,105].

Despite the compelling promise of miRNA-based therapy evident from basic research, translating it into clinical practice encounters considerable obstacles. The ability of miRNAs to target multiple genes, often viewed as a therapeutic advantage, also represents a dual-edged sword. Because a single miRNA can modulate numerous downstream mRNAs, altering its expression may simultaneously influence several signaling pathways. While effectively suppressing or activating disease-driving genes, this could inadvertently dysregulate genes essential for normal physiological functions, potentially leading to severe side effects or toxicity. Furthermore, existing therapeutic designs predominantly center on the canonical cytoplasmic roles of miRNAs, often overlooking the distinct roles of miRNAs localized to different subcellular compartments, which may lead to unintended consequences. For instance, in a porcine model of myocardial infarction, prolonged overexpression of miR-92a was found to induce aberrant cardiomyocyte proliferation and arrhythmia^[106]. As discussed earlier, nuclear-enriched miR-92a-3p enhances ANKRD1 transcription, leading to increased ANKRD1 protein, which in turn selectively upregulates MHC7 expression. This shift elevates the MHC7/MHC6 (β/α) ratio, ultimately impairing myocardial contractility and promoting hypertrophy and fibrosis^[38]. Given the faster contraction kinetics of MYH7 compared to MYH6, the physiological balance of these isoforms is essential for synchronous cardiac contraction^[107]. Disruption of this balance can cause regional mechanical asynchrony (e.g., regions contracting too rapidly or too slowly), triggering electrical instability and increasing arrhythmic risk^[107-109]. Thus, the arrhythmias observed following sustained miR-92a overexpression in pigs may reflect, at least in part, its nuclear activity, although the intrinsic pro-arrhythmic milieu of myocardial infarction cannot be ruled out as a contributing factor. Collectively, these findings highlight the necessity of considering miRNAs' subcellular localization and compartment-specific functions in the development of miRNA-targeted therapies and underscore the critical need to strictly control the duration and dosage of miRNA expression for clinical safety.

Biomarker-guided therapy facilitates therapeutic selection, efficacy assessment, dosage optimization, and the avoidance of side effects. Circulating miRNAs have emerged as a promising class of non-invasive biomarkers due to their unique physicochemical properties. Extracellular miRNAs, encapsulated in lipid vesicles or bound to carrier proteins/lipoproteins, are highly stable in biofluids, resistant to RNase degradation, and amenable to sensitive quantification via RT-qPCR and next-generation sequencing (NGS), offering practical advantages over conventional peptide markers^[110-114]. The expression of miRNAs is highly cell-specific and dynamically regulated by physiological states and pathological stimuli. These fluctuations provide a real-time molecular readout of cardiovascular status, underpinning their diagnostic and prognostic utility in cardiovascular diseases^[97,115,116]. For instance, circulating miR-126 levels correlate with platelet activation and are suppressed by aspirin, suggesting its role as a marker of antiplatelet efficacy^[117,118]. Patients with heart failure exhibit characteristic downregulation of 24 circulating miRNAs. Among these candidates, miR-26b-5p, miR-145-5p, miR-92a-3p, miR-30e-5p, and miR-29a-3p correlate with cardiac functional markers including LVEF and NT-proBNP, and their expression levels rise markedly in patients who achieve favorable responses to cardiac resynchronization therapy (CRT)^[119]. Levels of miR-1 and miR-133a directly reflect myocardial steatosis and are elevated in type 2 diabetes, consistent with altered myocardial lipid metabolism^[120]. In mouse models of acute myocardial infarction, cardiac-enriched miRNAs (miR-1, miR-133a, miR-208a, miR-499-5p) increase sharply in circulation and are trafficked to distant organs such as bone marrow, participating in systemic injury responses^[121]. In LVAD-supported patients, persistently elevated miR-483-3p tracks treatment response over time (at 3, 6, and 9 months post-LVAD support), while baseline miR-1202 outperforms NT-proBNP and INTERMACS scores in distinguishing responders from non-responders^[122]. Collectively, these findings highlight the great clinical potential of circulating miRNAs as biomarkers to track disease progression and predict therapeutic response as well as patients’ prognosis. However, these conclusions necessitate validation through large-scale studies with expanded cohorts.