The lethal-7 (let-7) microRNA (miRNA, miR), named because its knockout was lethal during development (1), was the first miRNA discovered in humans (2). It is well conserved across species (3), from Caenorhabditis elegans (C. elegans) to humans, even though higher animals tend to have increased numbers of let-7 isoforms. Both C. elegans and Drosophila melanogaster (D. melanogaster) possess a singular let-7 isoform (let-7a), while humans and mice both possess 10 isoforms (miR-98, miR-202, and let-7a, 7b, 7c, 7d, 7e, 7f, 7g, and 7i) across 12 and 3 genetic loci, respectively (4, 5). The let-7 isoforms (apart from miR-202) contain a conserved ‘seed sequence' with the nucleotides GAGGUAG in positions 2 to 8 of each miRNA molecule (5, 6). The preservation of this sequence between isoforms implicates similarly conserved biological targets and evolutionarily retained functions.

Expression of let-7 is ubiquitous in adult mammalian tissues and known to increase throughout development (7), but the specific expression levels of each let-7 isoform are context-dependent and tightly regulated (8). Due to its roles in developmental differentiation, proliferation, inflammation, and cellular metabolism, dysregulation of let-7 expression has been implicated in several pathologies, including cancers and cerebral and cardiovascular disorders (5).

In embryogenesis, let-7 plays a crucial role in controlling the development from the first trimester to birth (9–11). In the central nervous system (CNS), let-7 enhances glycogen biosynthesis by suppressing HMGA2 and NOTCH (12) and is implicated in maintaining endothelial cell integrity (3), thus providing an optimal functionality of the blood-brain barrier (13). Given its role as a regulator of angiogenesis, let-7 can also control cancer progression (14). Reduced let-7 expression is a common hallmark of most cancers (15), leading to the upregulation of genes critical for DNA replication, such as programmed cell death ligand 1 (PD-L1) (16) and high mobility group AT-hook 2 (HMGA2) (17). Additionally, apoptotic genes like caspase 3 (18), caspase 8, and B-cell CLL/lymphoma (BCL) (19), or the pluripotency transcription factors NANOG, SOX2, SOX9, OCT4, KLF4, and c-Myc are modulated by let-7 and help the survival of cancer stem cells (CSCs) (20, 21).

The let-7 family appears critical also for the progression of inflammation, with various members associated with early inflammatory pathways (5) through the well-established mechanism of action via the LIN28/let-7 axis. LIN28B has been identified as a molecular link between inflammation and cancer through a positive feedback loop involving NF-κB activation and the release of repression from the let-7-regulated IL-6 gene (22). More recent studies have associated the LIN28/let-7 axis with glucose metabolism because of its regulatory effects on PDK1, the function of which is to inhibit the activity of the pyruvate dehydrogenase (PDH) subunits of the pyruvate dehydrogenase complex (PDHC). Increased glucose metabolism is attributed to LIN28A/B-mediated repression of let-7 (23). In addition, in breast cancer, whilst LIN28A and LIN28B promote the oxidative metabolism of glucose, let-7, by acting on PDK1, promotes glycolytic consumption of glucose in a hypoxia-independent manner. This study suggests a direct regulation of the LIN28/let-7 axis in the glycolytic/oxidative modulation of glucose metabolism, even under normal oxygen levels (24). Inhibition of the enzymatic function of PDH shifts glucose-dependent energy production from mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis, thus acting as a gatekeeper of glucose metabolism. Increased activity in breast cancer stem cells (BCSCs) is also associated with elevated expression of the long non-coding RNA H19, which sequesters let-7 miRNA, leading to activation of HIF1a and enhancing downstream target PDK1 expression (25).

Furthermore, elevated expression of LIN28A/B enhances de novo fatty acid synthesis, promoting cancer progression via SREBP1. Lin28A/B directly associates with the mRNAs of both SREBP-1 and SCAP, enhancing the translation and maturation of SREBP-1, thereby accelerating metabolic conversion of saturated and unsaturated fatty acids and protecting cancer cells from lipotoxicity (26). A summary of the aforementioned functions of the let-7 family is presented in Table 1 and illustrated in Figure 1.

Despite the numerous evidence of let-7 implication in regulating the biological and metabolic functioning of nervous cells, there are no direct studies in models of neurodegeneration that have allowed us to establish whether its roles are neuroprotective or neurodegenerative. Most existing data link altered let-7 expression to neurological disorders such as multiple sclerosis, stroke, and Alzheimer's disease (27), while its role in traumatic brain injury (TBI) remains understudied. TBI is a leading cause of disability worldwide and the most frequent cause of death in individuals under 35 in Western countries (28).

Its global incidence is estimated at over 900 cases per 100,000 people annually, corresponding to nearly 69 million new cases each year. By 2030, the burden of TBI-related disability is expected to surpass that of Alzheimer's disease and cerebrovascular disorders, creating significant personal and socioeconomic costs (29, 30).

TBI is defined as a non-congenital injury to the brain caused by the nervous cell absorption of part of the external physical forces acting at the time of injury (primary insult), which induces the sudden alteration of a significant number of biochemical, metabolic, and molecular processes (secondary insult), modifying brain cell homeostasis and provoking transient or long-lasting (up to permanent) impairment of consciousness, neurocognitive deficits, neuromotor disabilities, or psychological disturbances. Grossly, TBIs are classified using the Glasgow Coma Scale (GCS), which categorizes TBI patients into mild (GCS range 13–15), moderate (GCS range 9–12), and severe (GCS range 3–8), based on a score obtained after specific neurological assessments, such as eye opening, motor, and verbal responses.

Despite this alarming epidemiological evidence, effective treatments remain scarce, and researchers are just beginning to unravel the complex cellular, molecular, and biochemical changes that influence TBI outcomes (31–35). It is now well accepted that the concept of the neurometabolic cascade involves a change in ionic cell homeostasis, mitochondrial dysfunction with energy penalty, sustained oxidative/nitrosative stress, glutamate excitotoxicity, glucose dysmetabolism, profound changes in protein and gene expressions, dangerous neuroinflammation, damage to the BBB permeability, and induction of apoptosis with a consequent significant increase in the nervous cell mortality rate (36, 37). The duration and the severity of the aforementioned neurochemical alterations are strictly dependent on the amount of energy absorbed at the time of impact and, ultimately, on the biomechanics of TBI.

Patients with mild (mTBI) injuries (including concussions, a peculiar type of mTBI frequently encountered in sports) often go undiagnosed because they are frequently characterized by rapidly resolving clinical symptoms. Notwithstanding, experimental and clinical studies have shown that mTBI and concussions provoke biochemical/metabolic/molecular changes in nervous cells lasting much longer than symptom resolution, which may have severe pathological and neurological consequences, particularly in the case of repeated injuries (38).

Diagnostic approaches for TBI rely on imaging and biomarkers. Although computed tomography (CT) remains the preferred method for assessing focal injuries, it lacks the sensitivity to effectively detect diffuse damage (39). Magnetic resonance imaging (MRI) offers greater resolution but is costly and less accessible (40). Among biochemical markers, proteins such as S100b, GFAP, and ubiquitin C-terminal hydrolase-1 (UCH-L1) (41, 42) have been widely studied, and the combination of GFAP and UCH-L1 has recently been FDA-approved for clinical use in evaluating mTBI (43). However, challenges in clinical translation persist, highlighting the need for novel biomarkers. Epigenetic mechanisms, including small non-coding RNAs (sncRNAs), DNA methylation, and histone modifications, have emerged as critical regulators of the post-injury response. miRNAs and snoRNAs are increasingly recognized as modulators of neuroinflammation and neuroprotection, while reciprocal regulation between miRNAs and epigenetic modifiers (e.g., DNA methyltransferases, histone deacetylases) suggests a dynamic network shaping long-term injury outcomes (44).

Within this context, several miRNAs, such as miR-21, miR-92a, miR-155, and members of the let-7 family, have shown altered expression following TBI and have been linked to processes including inflammation, metabolism, and neuronal survival (45). Importantly, miRNAs are highly stable in biological fluids and resistant to enzymatic degradation and freeze–thaw cycles, making them attractive candidates for non-invasive diagnostics (45). While emerging evidence supports their role as biomarkers, the contribution of let-7 specifically to TBI pathophysiology remains poorly defined. To clarify this relationship, the present review systematically evaluates the evidence for the differential expression of let-7 family members in TBI. It explores their potential role as both biomarkers and mechanistic mediators of injury outcomes.

To clarify current understanding, this systematic review examined the literature to answer the question: Which let-7 family members are differentially expressed in TBI?

This systematic review was registered with PROSPERO in 2025, CRD420251129282. Available from https://www.crd.york.ac.uk/PROSPERO/view/CRD420251129282.

This systematic review revealed the involvement of the let-7 family in the pathobiological mechanisms of TBI across varying severities and time points. Data from experimental TBI in laboratory animals and humans have notably highlighted that the different expression patterns depend on whether let-7 levels are measured in brain tissue (animal studies) or biofluids (animal and clinical studies).

Human studies showed an overall upregulation of let-7 family members across different biofluids, such as plasma, serum, and saliva, regardless of trauma severity (ranging from concussions to severe TBIs), population demographics (athletes vs. the general population), age, gender, or time since injury (from one day post-injury onward). The only exception was in CSF, where downregulation was predominant. In contrast, animal studies primarily demonstrated a downregulation of let-7 expression in brain tissue, with only a few exceptions. This pattern is not unique to TBI and aligns with findings in other diseases, such as cancer, where tissue levels of let-7 are generally low, but levels in the bloodstream are elevated, suggesting its potential as a biomarker (65–67). The inverse trend between tissue and biofluid levels may indicate a common regulatory mechanism for microRNAs, independent of the pathology considered, where cells release microRNAs to decrease their intracellular concentration, resulting in elevated levels in biofluids.

Based on the analysis of available literature, it can be stated that human studies have primarily focused on the use of let-7 as a biomarker for diagnostic and prognostic purposes. However, neither goal has been achieved to date, since (i) no correlation between let-7 levels in biofluids and TBI severity has been found, and (ii) no clear data have yet been produced correlating let-7 levels in biofluids with TBI patients' outcome.

Results of animal studies have explored more mechanistic aspects of the potential roles of let-7 variations in TBI. In the intricate landscape of one of the most complex and challenging pathologies affecting the brain, the let-7 family emerges as a crucial player in orchestrating the protective responses of nervous cells following TBI. Let-7 appears to play a crucial role in modulating the inflammatory cascade that typically follows TBI, which can both harm and help CNS recovery. Neuroinflammation is mainly driven by microglia and infiltrating macrophages, which amplify inflammation by releasing soluble cellular components in the injured brain (68, 69). Microglia, acting as the first responders to CNS injuries, can adopt either a “classically activated” M1 phenotype or an “alternatively activated” M2 phenotype (70, 71). While M1 polarization is associated with neuronal dysfunction and cell death, M2 polarization promotes anti-inflammatory responses, aids in debris clearance, and supports CNS repair. Modifying the M2/M1 ratio has shown promising therapeutic effects in TBI (72). MicroRNAs have been implicated in this modulation, with let-7c being one of the most abundant and conserved miRNAs linked to macrophage polarization (73, 74). Lv et al. (59) showed that let-7c was rapidly reduced in traumatically injured foci in the brains of adult C57BL/6J mice. The intracerebroventricular injection (ICV) of let-7c improved the outcomes of mice subjected to CCI by inhibiting neuroinflammation. In particular, the attenuation of microglia/macrophage activation was obtained through the inhibition of M1 polarization and enhancement of M2 polarization. In addition, authors showed that expressions of the let-7c putative target gene caspase-3 and of PKC-d (a modulator of caspase-3 effects) were inhibited. Miao et al. (60) also found that let-7c could be involved in the prevention and protection afforded by voluntary exercise in a mouse model of TBI induced by a pneumatic impact device. They showed that voluntary running wheel (RW) exercise prior to TBI significantly reduced the mortality rate and righting time in injured mice. They also showed that, following TBI, brain levels of let-7c were significantly different in the group of exercised mice compared to those of the untrained. Therefore, the authors proposed that an epigenetic mechanism, capable of inducing amelioration of functional deficits, might be involved in the beneficial effects of voluntary RW exercise following TBI.

It appears that let-7 can also help in minimizing TBI-associated secondary injury, particularly the neuroinflammatory processes caused by the release of pro-inflammatory cytokines (like TNF-α and IL-6), which can inadvertently cause further tissue damage. In this light, Johnson et al. used the rats' penetrating ballistic-like brain injury (PBBI) model to determine if changes in microRNA levels, including let-7i, were connected to those of neuroinflammatory mediators. Results showed an inverse correlation between let-7i and IL6, an interaction that was also previously described (75), suggesting let-7i as a plausible therapeutic target of TBI-induced inflammation. Hence, let-7 serves to mitigate the TBI-exacerbated neuroinflammatory response and directs immune cells toward protective functions. In this context, He et al. not only showed let-7i downregulation in a model of HSI in mice but also found that non-invasive intranasal delivery of let-7i significantly improves cognitive function and suppresses neuroinflammation, glial scar formation, and neuronal apoptosis via inhibition of the stimulator of interferon genes (STING). STING is a key regulator of inflammation, and its expression was found to be elevated in postmortem human TBI brains (76, 77). Balakathiresan et al. (63) and Sajja et al. (64) instead limited their studies to investigating microRNAs as potential biomarkers of TBI. They both found several members of the let-7 family differentially expressed in the CSF and serum of rodents undergoing blast injury.

However, since let-7′s roles are not limited to modulating mechanisms of inflammation, it is evident that the information obtained so far, in both experimental and clinical studies in TBI, is certainly not sufficient. For instance, several indications have been produced showing that the activity of the PDH complex, the key multi-enzymatic system ensuring the oxidation of pyruvate to form acetyl-CoA and thus fueling the Krebs cycle, is deeply modulated by let-7 through its action on the gene expressing the main PDH complex inhibitor (PDK, pyruvate dehydrogenase kinase). Using Marmarou's model of closed-head weight drop TBI in the rat, it was shown that profound glucose dysmetabolism (32) is associated with the PDH complex inhibition because of overexpression of PDK and concomitant downregulation of PDP (pyruvate dehydrogenase phosphorylase, the main activator of PDH) (78).

Additionally, although obtained by bioinformatic data only, it has also been indicated that let-7 may influence the homeostasis of N-acetylaspartate (NAA), the main neuron-specific N-acetylated amino acid deeply altered by TBI (79). Since the metabolism of NAA is strictly dependent on acetyl-CoA bioavailability (this compound is the acetyl group donor in the NAA synthesis catalyzed by NAT8L), it is easy to hypothesize that the let-7 modulation of both ASPA and PDK may have profound consequences on brain metabolism following TBI.

It should not, however, be disregarded that let-7 family members can influence neurogenesis. Particularly, the differentiation of neural stem cells into neurons in the TBI-injured brain may be highly relevant in aiding the replacement of lost or damaged cells after TBI. It should even be worth investigating whether they play a role in enhancing synaptic plasticity, which is crucial for functional recovery in cognitive tasks of the post-injured brain. Among the other potential roles of let-7 in the post-injury brain, the mitigation of oxidative/nitrosative stress, one of the mechanisms of nervous cell damage potently activated by TBI, might certainly be better characterized. Indications obtained under different pathological conditions, but TBI, suggest that let-7 is mutually regulated by FOXO3 and PRDX1 expression levels, both involved in the cellular capacity to maintain a correct oxido-reductive state of -SH groups of cysteine, deeply altered under conditions of oxidative/nitrosative stress (80). Also in this case, it has been shown that TBI deeply alters the cerebral concentrations of GSH, the main cellular antioxidant specific for keeping free -SH groups in the correct oxido-reductive state (32, 37), thereby rendering plausible the involvement of let-7 in the overall damaging process mediated by excess production of ROS and RNS occurring following TBI. Modulation of PI3K/Akt and MAPK pathways, thus promoting cell survival and growth, reinforces the brain's resilience in the wake of injury.

In the aftermath of TBI, the let-7 family serves as a multifaceted protector, navigating the complex interplay of inflammation, mitochondrial-related energy production, oxidative/nitrosative stress, and regeneration; each of them, if positively affected by let-7 modulation, may greatly improve nervous cell survival and, potentially, the outcome of TBI patients. Their contributions are vital for healing, illuminating pathways for future therapies that could help countless individuals reclaim their lives after the devastation of brain injury.

The present research highlighted the promise of let-7 as both a biomarker and a therapeutic target. Changes in its expression can indicate the severity of TBI, offering insights into patient prognosis. Moreover, the potential to manipulate let-7 levels, through mimics or inhibitors, opens new avenues for treatment, aiming to harness its protective capabilities and enhance recovery. However, the use of miRNA signatures as a novel diagnostic and prognostic tool remains largely in the descriptive phase yet. While a wealth of data has been gathered across various disease states, translating this information into clinical applications requires larger studies and universally accepted guidelines. One major issue is the need for standardized protocols for miRNA extraction, quantification, and analysis. Variability in sample collection, processing methods, and analytical techniques can lead to inconsistent results, hindering the establishment of universally accepted miRNA biomarkers. A shared information infrastructure for data exchange, analysis, and protocol standardization would greatly enhance research in miRNA biomarker discovery. Furthermore, the biological complexity of miRNA regulation necessitates a deeper understanding of their roles in specific contexts. For instance, miRNA expression can be influenced by factors such as age, gender, lifestyle, and environmental conditions. Therefore, identifying well-characterized endogenous miRNAs that are consistent across different populations and conditions is crucial for their clinical applicability.

Regarding animal models of TBI, these are essential for understanding the underlying mechanisms and potential treatments, yet the variety of methodologies and tissues analyzed can lead to discrepancies in results. Methodologically, injuries can be induced through various techniques, including controlled cortical impact, fluid percussion, weight drop, or blast overpressure, leading to differences in injury severity and patterns. Additionally, the specific tissues analyzed, ranging from the brain cortex to subcortical structures and the cerebrospinal fluid, can yield varying insights into the pathophysiological changes following TBI. For example, while some studies focus on acute inflammatory responses in the cortex, others might assess long-term neurodegenerative changes in deeper brain regions. This variability can complicate comparisons across studies and hinder the establishment of a consensus on the biological mechanisms involved in TBI, highlighting the need for standardized protocols and comprehensive analyses.

Importantly, our review is limited by the quality of available evidence. Of the 15 studies included, 9 showed an unclear or high risk of bias. This limitation significantly affects the strength of our conclusions and underscores the need for further high-quality, standardized, and rigorous research in this field. Establishing universally accepted guidelines and infrastructure for data sharing and analysis will be crucial to advancing miRNA-based diagnostics and therapeutics in TBI.

We found that the let-7 family of microRNAs plays a multifaceted role in the response to TBI. By regulating inflammation, promoting neuronal survival, enhancing neurogenesis, and modulating key signaling pathways, let-7 microRNAs play a crucial role in mitigating damage and facilitating recovery. Their potential as biomarkers and therapeutic targets highlights their importance in advancing TBI treatment strategies. miRNAs may soon become invaluable tools in clinical practice, revolutionizing the way we approach disease management and patient care. Further research into their specific mechanisms and effects may lead to innovative approaches for improving outcomes in TBI patients; however, such research is mandatory.