Ionizing radiation affects intracellular expression of miRNAs in various ways. Several studies have demonstrated the upregulation and downregulation of miRNAs following radiation exposure. For instance, in baboons, miR-212 exhibited 48- to 77-fold upregulation after 2.5 and 5 Gy irradiation, whereas miR-342-3p showed 10-fold downregulation (24,25). Similarly, radiation-induced miRNA regulation has been observed in clinical settings such as radiotherapy. In patients with breast cancer, radiotherapy results in a significant increase in miR-34a levels compared to non-treated or chemotherapy-treated patients (26), whereas miR-29a-3p and miR-150-5p levels gradually decrease with increasing doses of radiotherapy (27). These findings suggest that miRNA expression is influenced by radiation, with expression levels varying according to radiation dosage, cell type, and disease context. The regulation of miRNA expression following radiation exposure remains a complex and evolving field of research. Current research suggests that ionizing radiation alters miRNA expression through multiple mechanisms, including activation of the DNA damage response, modulation of the miRNA biogenesis machinery, and induction of epigenetic modifications. First, ionizing radiation induces the ataxia-telangiectasia mutated (ATM) kinase and activates p53, which, in turn, upregulates key miRNAs involved in apoptosis and DNA repair, including miR-34a and miR-16 (28). ATM-mediated phosphorylation of KH-type splicing regulatory protein (KSRP) enhances the processing of precursor miRNAs, such as let-7, increasing their levels post-radiation (29). Secondly, radiation influences the miRNA biogenesis machinery by modulating Drosha and Dicer, the key enzymes responsible for miRNA maturation. Studies have shown that Exportin-5, which transports precursor miRNAs to the cytoplasm, may also be affected by radiation-induced stress responses, further altering miRNA profiles (30). Finally, radiation-induced histone modifications and DNA methylation contribute to long-term changes in miRNA expression. For instance, radiation exposure has been linked to miR-21 upregulation, which enhances radioresistance by suppressing PTEN and activating PI3K/AKT signaling (31). Understanding these mechanisms will provide valuable insight into how miRNA regulation can be leveraged to enhance radiosensitivity in cancer therapies. However, the precise mechanisms by which ionizing radiation regulates miRNA expression remain largely unknown. Ongoing research is aimed at elucidating these mechanisms and improving our understanding of the interaction between radiation and miRNA regulation.

Although the mechanistic foundation of miRNA regulation by radiation remains unclear, efforts have been made to explore how radiation influences miRNA biogenesis and how radiation-induced miRNAs affect cellular responses to radiation. Proposed mechanisms linking the radiation response to enhanced miRNA biogenesis include increased processing of pri-miRNAs via KSRP following phosphorylation by the DNA damage sensor protein ATM as well as the activation of pri-miRNA transcription driven by the DNA damage-stabilized transcription factor p53 (32,33).

Tumor radiosensitivity is a critical determinant of radiotherapy outcomes and miRNAs play a significant role in modulating various aspects of tumor radiosensitivity by regulating key cellular processes such as cell cycle arrest, DNA damage repair, cell death, and radiation-related signal transduction (34). miRNAs can hinder the production of proteins that are crucial for recognizing DNA damage. They can also interfere with signaling pathways and cell cycle arrest mechanisms, thereby initiating repair processes. This can lead to a diminished DNA repair capacity and increased radiosensitivity. For instance, miR-24 and miR-451 suppress the expression of DNA damage sensor proteins H2AX and ATM, respectively (35,36). Both miRNAs influence cell cycle progression during stress responses. miR-421 targets ATM, impairing S-phase cell cycle arrest, whereas miR-24 regulates cell cycle proteins, such as cyclins A and E, as well as the retinoblastoma protein. miRNAs can also directly target DNA repair pathways. For example, miR-210 represses RAD52, a protein involved in homologous recombination, whereas miR-101 regulates DNA-PK kinase, which is crucial for nonhomologous end joining (37,38). Moreover, miRNAs can modulate the downstream signaling pathways involved in the radiation response, which ultimately influences cell survival or death. This includes pathways such as PI3K/AKT, nuclear factor-kappa B, mitogen-activated protein kinase, and transforming growth factor-beta. For instance, a suppressive effect of miR-221 and miR-222 on the AKT pathway by targeting the upstream tumor suppressor PTEN has been observed in gastric carcinoma cells, where they promote increased cell death and radiosensitivity (39). Since miR-210, which was previously discussed, is hyperactivated in hypoxic environments and controls tumor survival, it is especially notable that miRNAs may potentially influence external stimuli like radiation (33,38,40). Given that radiotherapy consistently involves radiation exposure, albeit at varying doses, it is reasonable to conclude that miRNAs are important determinants of the tumor response to radiotherapy (Table II).

The functions of miRNAs in radiotherapy vary significantly depending on the type of cancer, with miRNAs being either upregulated or downregulated to act as oncogenes or tumor suppressors. In breast cancer, miR-144 is upregulated following radiotherapy and contributes to radiation resistance by inhibiting radiation-induced apoptosis in cancer cells. A study analyzing 207 early invasive breast cancer samples with a 10-year follow-up validated these findings using over 1,000 cases and identified key miRNAs associated with radiotherapy resistance (41). Yu et al (41) demonstrated that miR-144 inhibits caspase-3 and caspase-7, preventing these caspases from initiating radiation-induced apoptosis. Consistent with this finding, miR-144 overexpression has been associated with poor prognosis in patients with breast cancer (42). In contrast, miR-200c has been identified as a radiosensitizing miRNA in breast cancer radiotherapy, enhancing ionizing radiation-induced double-stranded breaks and apoptosis by directly inhibiting KRAS in breast cancer cell lines. Notably, the expression of miR-200c increases in a dose-dependent manner upon radiation exposure, and its overexpression varies depending on the type of breast cancer (43,44). Another key miRNA is let-7d, and although it did not exhibit changes in expression levels during radiotherapy, it plays a critical role in sensitizing triple-negative breast cancer stem cells to radiation by repressing their self-renewal capacity. The combination of radiotherapy and let-7d significantly inhibited tumor growth by targeting the cyclin D1/Akt1/Wnt1 signaling pathway, which is crucial for breast cancer treatment (45,46). Additionally, miR-139-5p expression was significantly increased in patients undergoing radiotherapy who experienced favorable clinical outcomes, including no relapse or death. Research has demonstrated that miR-139-5p plays a key role in enhancing radiosensitivity by inhibiting DNA repair and reactive oxygen species defense mechanisms, ultimately inducing apoptosis in irradiated breast cancer cells (47).

In summary, miRNAs such as miR-144, miR-200c, let-7d, and miR-139-5p exhibit distinct roles in modulating the breast cancer response to radiotherapy, either promoting radioresistance or enhancing radiosensitivity. These findings underscore the potential of miRNA-based therapies to improve the efficacy of radiotherapy in patients with breast cancer.

Radiotherapy is a conventional treatment option for localized prostate cancer with overall survival rates comparable to those of radical prostatectomies. Several miRNAs have been shown to modulate prostate cancer cell responses to radiation, either by enhancing radiosensitivity or promoting radioresistance (48). miR-145 is upregulated in most prostate cancer cells after 4 h of irradiation. miR-145 suppresses DNMT3b expression by directly targeting the 3′ UTR of DNMT3b mRNA, which sensitizes prostate cancer cells to radiation by reducing DNMT3b levels (49). In contrast, miR-95 is upregulated in prostate cancer cells after irradiation and mediates radiotherapy resistance by downregulating SGPP1, a gene that promotes cell death and activates the S1P-PI3K-AKT survival pathway. Elevated miR-95 expression correlates with more aggressive prostate cancer phenotypes and poor prognosis, as demonstrated by next-generation sequencing and functional validation using prostate cancer cell lines and mouse xenograft models. The study further confirmed SGPP1 as a direct target of miR-95 and identified enhanced cell proliferation and impaired G2-M checkpoint as key mechanisms of resistance (50,51). miR-9, another miRNA upregulated during radiotherapy, inhibits tumor growth by suppressing MEKK3 protein expression (52,53). Additionally, miR-22 and miR-30a are upregulated in irradiated prostate cancer cells and enhance radiosensitivity by modulating ATP citrate lyase and TP53INP1, which influence metastasis and autophagy in prostate cancer (CaP) cells (54,55). Several miRNAs were modulated at 24 h after radiation exposure. miR-106b is downregulated in irradiated prostate cancer cells, and its downregulation is associated with improved prognosis, as it prevents miR-106b from overriding radiation- or therapy-induced p21 activation, thereby preventing radioresistance. Similarly, miR-521, which induces radiosensitivity by inhibiting the DNA repair protein Cockayne syndrome protein A, is downregulated and contributes to radioresistance in CaP cells (56). Conversely, miR-449a is upregulated and enhances radiosensitivity by reducing c-Myc transcription in CaP cells (57). The let-7 family and miR-34a are typically downregulated in prostate cancer but are upregulated in certain radiosensitive CaP cell lines following fractionated radiation exposure (52). Let-7 regulates prostate cell homeostasis by targeting RAS, c-Myc, HMG family proteins and androgen receptors (58). miR-34a inhibits cancer stemness and targets CD44, MET, BCL2, and SIRT1, which interact with p53 to suppress tumorigenesis. Clinically, upregulated miR-34a expression is associated with a better prognosis in patients with prostate cancer (59,60). Another important miRNA in prostate cancer prognosis is miR-200a, whose downregulation has been linked to poor outcomes in prostate cancer (61). Similarly, miR-200b and miR-200c act as tumor suppressors in prostate cancer, and their downregulation leads to chemoresistance and radioresistance in tumor cells (62). Several miRNAs, such as miR-126, miR-20b, miR-203, let-7g, miR-30b, miR-30a, and others, are downregulated in LNCaP prostate cancer cells following irradiation. These miRNAs, which modulate radiosensitivity, were not consistently downregulated across studies and their expression varied depending on radiation dosage and timing (54,63–65).

Despite the increasing number of miRNAs involved in radiotherapy, most studies on prostate cancer have been conducted in vitro. Therefore, further research using animal models and human clinical trials is required to better understand the role of miRNAs in radiation therapy for prostate cancer.

Radiotherapy is primarily used to treat non-small cell lung cancer (NSCLC), which accounts for approximately 85% of all lung cancers. Many miRNAs are either upregulated or downregulated following radiation exposure, with some contributing to radiosensitivity and radioresistance. Among the miRNAs overexpressed in radiosensitive cancers are miR-126, miR-451, miR-128b, miR-let-7a, and miR-495, whereas miR-15b, miR-22, miR-106b, miR-19b, miR-21, miR-17-5p, and miR-130a are downregulated compared to their levels in radioresistant cancers. miR-126 enhances the sensitivity of NSCLC cells to radiation by regulating the PI3K-Akt pathway. One study analyzed miRNA expression in 30 patients with NSCLC undergoing postoperative radiotherapy and identified 12 differentially expressed miRNAs (66). miR-451 increases radiosensitivity by promoting irradiation-induced apoptosis via the PTEN pathway. In another study, pre-miR-451 was transfected into A549 NSCLC cells and its effects were analyzed using clonogenic assays, apoptosis analyses, and western blotting. Irradiation (0–6 Gy) showed that miR-451 overexpression enhanced apoptosis and increased radiosensitivity via PTEN activation (67). miR-128b acts as a tumor suppressor by suppressing the mRNA expression of epidermal growth factor receptor (EGFR) and subsequently reducing tumor growth. In another study, miR-128-b and EGFR expression were analyzed in 42 NSCLC patient samples using RT-qPCR and immunohistochemistry. A549 lung cancer cells transfected with miR-128-b mimics and inhibitors revealed a negative correlation between miR-128-b and EGFR expression in NSCLC (68). miRNA-let-7a inhibits cell growth by targeting cyclin D1-associated factors, thereby reducing the migration and invasion of tumor cells. Let-7a expression is also positively associated with the efficacy of radiotherapy in patients with lung cancer with brain metastasis (66,69,70). miR-495 functions as a tumor suppressor by inhibiting tumor progression via targeting TCF4 expression and repressing epithelial-mesenchymal transition (EMT) and the Wnt/β-catenin pathway. In radiotherapy, miR-495 reduces the radiation-induced bystander effect, improves patient tolerance to radiotherapy, and enhances clinical outcomes (71–73). Conversely, miR-15b protects cells from radiation-induced stress by promoting p53 phosphorylation and facilitating DNA repair. While this function is oncogenic in NSCLC, it helps protect the surrounding lung cells from the damaging effects of radiation (74,75). Interestingly, miR-22 acts as a tumor suppressor by inhibiting angiogenesis by targeting SIRT1 and FGFR1, but is downregulated in radiosensitive NSCLC (76). In contrast, miR-22 is upregulated in small cell lung cancer (SCLC) during γ-irradiation, where it enhances radiosensitivity by targeting WRNIP1 (77). miR-106b plays a key role in tumor formation by suppressing BTG3 and is associated with resistance to both chemotherapy and radiotherapy (78,79). miR-19b is a critical biomarker in lung cancer and is known to enhance cell proliferation and resistance to apoptosis, drugs, and radiation by modulating EGFR signaling (80,81). miR-21, a well-known miRNA implicated in various cancers, promotes growth, metastasis, and resistance to chemotherapy and radiotherapy in NSCLC by targeting PTEN. Silencing miR-21 expression promotes radiosensitivity in lung cancer (82,83). miR-17-5p negatively regulates TBP2, a tumor suppressor gene, in lung cancer, and its silencing reduces cell viability, invasion, migration, and resistance to therapy (84). One study found that miR-130a plays a complex role, as its upregulation has been associated with low survival rates in patients with NSCLC after radiotherapy, although its exact function remains unclear. While miR-25 and miR-191 are associated with poor survival outcomes, miR-130a is both an oncogene and a tumor suppressor, depending on the context. Low miR-130a expression can lead to poor survival in patients with NSCLC by inhibiting KLF3, a key regulator of lung cancer growth (85,86). Finally, although not downregulated, miR-410 is upregulated in most NSCLC cases and has been found to promotes radioresistance by binding to PTEN and indirectly activating the PI3K/mTOR pathway, highlighting its role in therapeutic resistance (87).

In summary, miRNAs play diverse roles in regulating radiosensitivity and radioresistance in NSCLC, with some acting as tumor suppressors and others as oncogenes. Continued research on miRNA-targeted therapies holds promise for improving the efficacy of radiotherapy in lung cancer treatment.

Nasopharyngeal cancer (NPC) is predominantly characterized by poorly differentiated and undifferentiated squamous cell carcinomas. Due to the unique anatomical location and locally invasive growth pattern of NPC, surgical intervention is often unsuitable, making radiotherapy the most effective treatment option. Several miRNAs have been implicated in promoting radioresistance in NPC. miR-19b-3p, miR-125b, miR-21, and miR-205 contribute to NPC recurrence by enhancing radiotherapy resistance through the regulation of BCL2 family proteins (88). In contrast, miR-203 is upregulated in radiosensitive NPC, exerting its effect by downregulating IL8/AKT signaling (89). miR-222 is commonly upregulated in NPC cell lines and promotes radioresistance by targeting PTEN (90). Furthermore, miR-9 expression increases following radiation exposure and suppresses apoptosis in NPC cells by modulating glutathione levels (91). In contrast, miR-120 is down-regulated in most NPC cell lines, reducing their sensitivity to radiation. Studies have shown that miR-120 upregulation decreases the survival fraction of NPC cells by targeting PDCD6, inhibiting BCL2, and activating caspase-3 and histone H2AX phosphorylation (92).

Although radiotherapy is not typically the first-line treatment for gastric cancer (GC), recent studies have demonstrated its benefits in locally advanced gastric cancer, showing a reduction in both mortality and recurrence compared with surgery alone (93). Several miRNAs have been implicated in the response of gastric cancer to radiotherapy. miR-21, miR-24, miR-421, and miR-605 are upregulated in diffuse-type GC and play oncogenic roles by increasing radioresistance by targeting ATM/ATR/H2AX, thereby impairing DNA damage repair (94). Additionally, miR-192 and miR-215 are naturally upregulated in most GCs, promoting tumorigenesis by activating the Wnt/β-catenin pathway through the targeting of APC, which in turn enhances DNA damage repair (95). In contrast, miR-129-5p functions as a tumor suppressor by inhibiting nucleolar and spindle-associated protein 1 (NUSAP1). The overexpression of NUSAP1 in GC leads to increased radioresistance and enhanced DNA damage repair, resulting in poor patient prognosis (96). Similarly, miR-4537 acts as a tumor suppressor by binding to ZNF587 and suppressing its expression. In GC cells, miR-4537 inhibits cell proliferation while enhancing apoptosis and increasing radiosensitivity (97). miR-4766-5p is another tumor suppressor that is typically downregulated in GC cells, where it inhibits cancer progression by targeting NKAP and inactivating the AKT/mTOR pathway, thereby promoting radiosensitivity in GC (98). In addition, miR-300 and miR-642 function as tumor suppressors by modulating apoptosis and cell cycle regulation. These miRNAs are downregulated in GC cells after radiation, and although their target genes and pathways remain under investigation, they have been shown to increase apoptosis and reduce DNA damage repair in GC cells. Intriguingly, some studies have associated miR-300 and miR-642 with chemoresistance in other tumor cell lines, suggesting that they may serve as promising biomarkers for the treatment, diagnosis, and prognosis of GC (99). In summary, miRNAs play diverse roles in the response to radiotherapy in both nasopharyngeal and gastric cancers, thereby influencing radiosensitivity and resistance. Further research on the roles of specific miRNAs could provide valuable insights for improving the therapeutic outcomes of these cancers.

Radiotherapy is typically the first-line treatment for brain tumors, particularly localized brain tumors. In response to radiotherapy, 16 miRNAs were found to be downregulated: miR-5687, miR-4766-3p, miR-4690-3p, miR-4262, miR-302d-3p, miR-6752-5p, miR-548ao-5p, miR-4772-3p, miR-485-5p, miR-511-5p, miR-1471, miR-2276-5p, miR-548n, miR-3132, miR-425-3p, miR-4460, miR-4262, and miR-302d-3p. In contrast, 19 miRNAs were upregulated, including miR-7153-3p, miR-609, miR-373-5p, miR-5582-3p, miR-4662a-3p, miR-619-5p, miR-3656, miR-502-5p, miR-6754-3p, miR-4804-3p, miR-3199, miR-4434, miR-3677-5p, miR-4528, miR-4731-5p, miR-144-3p, miR-548×-3p, miR-4795-5p, miR-1276, miR-502-5p, and miR-144-3p (100). miR-4262 has been implicated in promoting cell proliferation and migration in gliomas by targeting large tumor suppressor 1 (101). Similarly, miR-302d-3p enhanced cancer cell proliferation by influencing various biological pathways. Radiotherapy may reduce brain cancer cell proliferation by downregulating miR-4262 and miR-302d-3p, thereby potentially inhibiting tumor growth (101). Although the exact mechanisms underlying the upregulation of miR-502-5p and miR-144-3p in brain tumors remain unclear, both miRNAs have shown tumor-suppressive activity in other cancers. Their expression is associated with a better prognosis in glioblastoma, suggesting that they may act as potential tumor suppressors in brain cancer. Consistently, the upregulation of miR-502-5p and miR-144-3p has been linked to decreased cell proliferation in glioma, further supporting their role as tumor suppressors (100,102).

Radiotherapy, often combined with cisplatin, is the first-line treatment for cervical cancer. Numerous proteins involved in radiosensitivity are regulated by miRNAs, and approximately ten miRNAs have been identified as key regulators of radiosensitivity in cervical cancer. Among these, miR-9, miR-21, miR-200a, miR-218, miR-34a, miR-23b, and miR-203 have been shown to enhance radiosensitivity, whereas miR-421, miR-181a, and miR-106b promote radioresistance (103). miR-9 enhances radiosensitivity and inhibits angiogenesis by targeting suppressor of cytokine signaling 5, a key protein involved in cytokine regulation (104). miR-21, a well-studied miRNA in various cancers, functions as a tumor suppressor in cervical cancer by downregulating the RECK signaling pathway and inhibiting cancer growth and migration. However, the role of miR-21 is controversial because it represses PTEN and contributes to drug resistance in cancer cells (105,106). Low expression of miR-200a in cervical cancer has been linked to pronounced radioresistance, whereas miR-218 and miR-34a enhance radiosensitivity by promoting radiation-induced apoptosis (107–109). miR-23b increases radiosensitivity and chemosensitivity by suppressing the biological progression of cervical cancer through direct targeting of Six1, as well as by modulating EMT and the AKT/mTOR signaling pathway (110,111). miR-203 inhibits cervical cancer cell growth by inducing cell cycle arrest and apoptosis, thereby contributing to its radiosensitizing effects (112). In contrast, miR-421 promotes radioresistance by regulating ATM, a key sensor in the ionizing radiation response, which functions alongside the ATR and BRCA proteins (113). miR-181a and miR-106b contribute to radioresistance by inhibiting immediate early response 3, a regulator of apoptosis (106). Additionally, other miRNAs, such as miR-630, miR-1246, and miR-1290, have been reported to be upregulated in radiation-resistant cervical cancer. The upregulation of these miRNAs after irradiation has been associated with enhanced cancer cell survival, highlighting their potential role in promoting radioresistance (114–116).

Despite advancements in miRNA-based therapeutics, clinical translation is hindered by challenges related to the immune response, delivery efficiency, and targeting specificity. Naked miRNA molecules and virus-based delivery vectors can be recognized as foreign agents that trigger immune activation, which may cause systemic toxicity. This issue was evident in a phase I clinical trial of MRX34, a miR-34a mimic, which was halted because of severe immune-related adverse effects (117). To mitigate immunogenicity, researchers have explored chemical modifications, such as 2′-O-methylation and locked nucleic acids, which enhance stability and reduce immune activation. Additionally, exosome-based miRNA delivery systems have shown promise because of their natural biocompatibility and ability to evade immune detection. Early research on miRNA delivery primarily relied on viral vectors; however, their clinical application was limited due to significant safety concerns. Polylactide-co-glycolide (PLGA) particles, widely used for RNA transport, are considered potential miRNA delivery vehicles, but their low efficiency has proven insufficient for clinical application. Among the lipid-based delivery systems, neutral liquid emulsions (NLEs) were initially explored because of their low toxicity; however, their delivery efficiency remains suboptimal (118,119). Currently, safer and more effective delivery methods are being investigated. TargomiR, a delivery system utilizing bacterially derived mini-cells containing miRNA mimics and targeting moieties, has shown encouraging results in mesothelioma and NSCLC. Phase I trials have demonstrated significant suppression of target miRNAs, indicating their potential for cancer treatment. Another promising approach is the use of neutral liposomes, such as 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), which has been widely used in small interfering RNA (siRNA) delivery. Preclinical studies using DOPC liposomes loaded with miRNA-506 mimics or miRNA-520 demonstrated significant tumor suppression in ovarian cancer models, whereas miR-2000-complexed DOPC liposomes effectively inhibited tumor growth in lung cancer models (118,119). Additionally, targeting specificity remains a significant challenge in miRNA-based therapies to ensure precise delivery to tumor cells while minimizing unintended effects on normal tissues. Ligand-conjugated nanoparticles such as antibody-functionalized liposomes or aptamer-based delivery systems enhance specificity by targeting tumor-associated antigens. Moreover, CRISPR/Cas-mediated miRNA editing offers a precise approach for modulating miRNA activity in cancer cells, thereby reducing systemic toxicity and off-target effects (120). Alternative polymer-based delivery methods have also been investigated. Early research on polyethyleneimines (PEIs) revealed high cytotoxicity and low transfection efficiencies, limiting their clinical use. However, modifications such as polyethylene glycol (PEG) conjugation have significantly improved biocompatibility and delivery efficiency. Similarly, chitosan, a cationic polymer derived from chitin, has been studied as a biocompatible alternative for miRNA delivery with promising results in preclinical models (110).

miRNAs have emerged as promising noninvasive biomarkers for cancer detection and prognosis because of their stability in bodily fluids and their disease-specific expression profiles. However, their integration into routine clinical practice requires overcoming the challenges related to the standardization of detection protocols, quality control, and integration with traditional biomarkers. Currently, multiple detection methods, including quantitative real-time PCR, droplet digital PCR, and next-generation sequencing, are used for miRNA profiling. However, variability in sample collection, RNA extraction, and normalization complicates its clinical implementation. Establishing standardized protocols using reference miRNAs, such as miR-16 or let-7a, is crucial for improving reproducibility. Moreover, ensuring quality control and validation is vital, as pre-analytical factors, such as hemolysis in blood samples and RNA degradation, can introduce variability in miRNA measurements. The incorporation of spike-in-control RNAs and stringent quality control procedures can enhance the reliability of miRNA-based diagnostics. Although miRNA profiling alone provides valuable insights, its clinical utility can be amplified through multi-marker integration. For instance, combining miR-21 expression analysis with PSA testing has demonstrated improved accuracy in the diagnosis of prostate cancer (3). Similarly, artificial intelligence-driven models are being developed to analyze miRNA signatures and conventional clinical parameters, thereby fostering precision medicine approaches for personalized cancer treatment. With the evolution of miRNA-based therapeutics and diagnostics, key research directions must be prioritized. Optimization of multifunctional miRNA delivery platforms, integration of tumor-targeting peptides, exosome-based carriers, and CRISPR-based precision editing could enhance therapeutic specificity while minimizing off-target effects. Further advancements in miRNA-based liquid biopsy will facilitate early cancer detection and monitoring. Additionally, research on the stability of miRNAs in body fluids and storage conditions will improve their reliability as diagnostic biomarkers. Leveraging machine learning algorithms trained on large miRNA datasets can enhance diagnostic precision and enable tailored treatment strategies for individual patients. Another promising area of research involves anti-angiogenic miRNAs, which have the potential to improve radiotherapy efficacy by normalizing the tumor vasculature and reducing hypoxia-related resistance. For example, miR-210 regulates angiogenesis through the vascular endothelial growth factor (VEGF) signaling pathway, and its inhibition can restore normal blood vessel function and enhance oxygenation and radiosensitivity in tumors (121–123). Similarly, inhibiting miR-155, which promotes neovascularization by modulating the ELK3 and E2F2 transcription factors, could enhance radiosensitivity and reduce tumor progression (113). miRNA-based interventions have the potential to revolutionize cancer management by addressing these challenges and refining miRNA detection methodologies to ensure greater efficacy and safety in therapeutic and diagnostic applications.

In 2016, the biotechnology company miRNA Therapeutics, now known as Synlogic, discontinued phase 1 clinical trials of the miRNA-34 mimic drug MRX34, intended for cancer treatment, after five patients experienced severe immune reactions resulting in serious adverse events (SAEs). Consequently, the planned phase 2 trials of MRX34 for melanoma were also canceled. Since then, no phase 3 trials have been registered at clinicaltrials.gov, although ongoing reviews and new strategies for utilizing miRNAs as therapeutic agents are in progress. Early studies on miRNA delivery relied primarily on viral vectors. However, as demonstrated by the adverse events mentioned above, viral vectors pose significant safety concerns in clinical use. PLGA particles, which are widely used to transport small RNAs, have been considered as potential miRNA delivery vehicles, but the low efficiency of miRNA loading has proven insufficient for clinical application. Among lipid-based delivery systems, NLEs have been extensively tested because of their low toxicity; however, they are not sufficiently efficient for miRNA delivery. Despite these initial challenges, safer and more effective delivery methods are currently being investigated. One promising technology is TargomiR, a delivery system that uses bacterially derived mini-cells containing miRNA mimics and targeting moieties (antibodies that recognize proteins on target cells). The use of TargomiR has shown encouraging results in mesothelioma and NSCLC, with phase 1 trials demonstrating significant suppression of target miRNAs, suggesting a viable method for treating these cancers (117). Additionally, neutral liposomes such as DOPC are commonly used for siRNA delivery and are currently being tested for miRNA delivery in preclinical studies. For example, DOPC liposomes loaded with miRNA-506 mimics or miRNA-520 were shown to significantly suppress tumor growth in an ovarian cancer orthotopic mouse model. Similarly, DOPC liposomes complexed with miR-2000 effectively inhibited tumor growth in an orthotopic lung cancer model. Polymeric delivery methods such as those utilizing PEIs initially exhibit low transfection efficiencies and high cytotoxicity. However, alternative polymers, such as PEG, have emerged as more favorable miRNA delivery vehicles owing to their improved biocompatibility and lower toxicity. PEG can be covalently fused to PEI to enhance its stability and efficiency. Chitosan, a cationic polymer derived from chitin, has been studied as a biocompatible natural polymer for miRNA delivery (118,119). Ongoing studies are exploring novel delivery systems for miRNA therapeutics, as outlined in Table III.

Although there is great potential for miRNA therapeutics, challenges remain, including the pleiotropic nature of miRNAs and the complexity of their interactions with cellular processes and proteins. These factors underscore the need for further clinical and bioinformatics research to support reliable clinical applications (120). Nevertheless, the development of sophisticated miRNA delivery systems could unlock diverse therapeutic and clinical applications, particularly for enhancing the effectiveness of radiotherapy by increasing the radiosensitivity of cancer cells. The miRNAs listed in Table IV, which are associated with favorable clinical outcomes, emphasize the importance of low toxicity and high specificity in miRNA therapeutics. Moreover, anti-angiogenic miRNAs have the potential to reduce the side effects and increase the effectiveness of radiotherapy. Recent preclinical and clinical trials combining radiotherapy with anti-angiogenic drugs have shown promising results. The enhancement of radiotherapy through these drugs may be attributed to improved tumor oxygenation stemming from the normalization of blood vessels and suppression of angiogenic growth factors typically stimulated by radiotherapy. However, these drugs exhibit unnecessary toxicity and adverse effects in patients, and rapid resistance to current anti-angiogenic therapies is becoming a concern. As alternative strategies to target angiogenesis are required, miRNAs have emerged as potential antiangiogenic agents. For example, miR-210 induces angiogenesis by mediating the VEGF signaling pathway. The inhibition of miR-210 in hypoxic tumors can restore abnormal tumor vasculature, improve oxygenation, and sensitize tumors to radiotherapy (124,125). Similarly, inhibition of miR-155 can enhance radiosensitivity because miR-155 regulates angiogenesis and promotes neovascularization by modulating the transcription of ELK3 and E2F2 (121,122).