The role of microRNAs in modulating Wnt signaling pathway dynamics and their therapeutic implications in non-small cell lung cancer

Abstract

Lung cancer ranks as the second most prevalent cancer worldwide and exhibits the highest mortality rate among all cancers. It primarily consists of two subtypes: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with NSCLC representing 80–85% of cases. The Wnt/β-catenin signaling pathway is crucial in lung cancer initiation, progression, metastasis, and chemoresistance. MicroRNAs (miRNAs), small non-coding RNAs, significantly influence the tumor microenvironment by modulating cancer cell proliferation, angiogenesis, and apoptosis through the targeting of specific genes. Depending on their targets, miRNAs can function as oncogenes or tumor suppressors, highlighting their potential as therapeutic targets. Additionally, miRNAs serve as novel biomarkers for cancer diagnosis. Understanding the key miRNAs involved in lung cancer and their interactions with pathways like Wnt is essential for developing new diagnostic and therapeutic approaches. This review focuses on how specific miRNAs affect the Wnt pathway's components and their roles in tumorigenesis, metastasis, and therapy resistance, emphasizing their potential as therapeutic targets in NSCLC. Ultimately, this review aims to improve clinical outcomes and facilitate personalized therapeutic approaches for this aggressive malignancy.

Graphical Abstract

Introduction

Lung cancer (LC) is reported as the second most common cancer and has the highest mortality rate among different types of cancers [1, 2]. Currently, the five-year survival rate for patients with LC is merely 19%, with 57% diagnosed at an advanced stage, resulting in a 5-year survival rate of only 5% [3, 4]. Pathologically, LC has two main types: Small Cell Lung Cancer (SCLC) and Non-SCLC (NSCLC). SCLC is more metastatic and accounts for about 15% of LC cases, while NSCLC accounts for nearly 85% [5]. NSCLC is further classified into three main subtypes, including lung adenocarcinoma (approximately 40% of cases), Squamous cell carcinoma (25–30% of cases), and large cell carcinoma.

Various risk factors for LC include environmental and occupational exposures, such as asbestos, radon or urban air pollution, chronic lung disease, lung infections, and lifestyle factors. However, the majority of LC cases (85%) are attributed to tobacco smoking, and people who do not smoke may still suffer LC due to becoming second-hand smokers [6, 7]. Due to the molecular complexity of LC and the presence of drug resistance, the rate of treatment failure is significantly high [8].

In human malignancies, deregulation of Wnt/β-catenin pathway is prominent [9–12]. These findings provide evidence that Wnt/β-catenin deregulation could be a cause of initiation and progression of various cancers including colorectal cancer, cholangiocarcinoma, pancreatic ductal adenocarcinoma (PDAC), leukemia, melanoma, and breast cancer [13]. Abnormal upregulation of the pathway can affect stem cell fate, stimulate tumorigenesis, and facilitate tumor development, metastatic ability, and drug resistance to radiotherapy, chemotherapy, immunotherapy, and targeted therapy [14, 15]. LC can develop following abnormal regulation of various signaling pathways, including the Wnt/β-catenin pathway. Although the most related malignancy caused by the Wnt signaling pathway is colorectal cancer, recent investigations support a potentially significant role for Wnt pathway components in LC initiation and progression [16]. For example, Wnt pathway genes are overexpressed in the lungs of Kras mutant mice with stimulated tumorigenesis [17]. In addition, a study has shown that β-catenin expression might repress antitumor activity by down regulating tumor-infiltrating CD8 + T cells. Suppression of β-catenin expression and/or function might be beneficial for anti-cancer immunotherapy [18]. Overally, based on preclinical studies conducted in recent years, The targeted approach to the Wnt/β-catenin pathway has demonstrated remarkable effectiveness in combating NSCLC, and various Wnt pathway inhibitors have great potential for combination therapy in NSCLC [19, 20]. Consequently, numerous anti-LC drugs have been formulated to inhibit the Wnt signaling pathway [21].

MicroRNAs (miRNAs) regulate signaling pathways by inhibiting the translation of genes that play a key role in regulating those pathways [22, 23]. Deregulated Wnt pathways initiate and progress various human malignancies. In the canonical pathway, β-catenin builds up within the cell and then translocates to the nucleus to regulate the expression of target genes. In contrast, the non-canonical pathway operates without the involvement of β-catenin within its functioning [24]. Wnt signaling regulates differentiation and cell fate in early embryogenesis through various components that contribute to the developing lung. Aberrant expression of Wnt/β-catenin-responsive genes promotes NSCLC progression. In LC, the signaling of the Wnt/β-catenin pathway may be deregulated in various aspects, such as Wnt ligand, Frizzled (FZD) receptor, T-cell factor (TCF)/lymphoid enhancer factor (LEF)-dependent transcription, and WNT suppressor silencing, affecting the appearance and development of LC [21, 25]. The present review aims to collect the miRNAs regulating LC by influencing the Wnt signaling pathway and then introduce their limitation and therapeutic potential in LC management.

The overview of the canonical Wnt signaling pathway

Wnt signaling has been reported as an important player in several biological processes, and its components serve as valuable biomarkers and potential targets for cancer treatment [26]. Wnt ligands are hydrophobic polypeptides consisting of approximately 350 residues that produce diverse signals, ultimately stimulating the TCF/LEF family of transcription factors. The Wnt/β-catenin signaling pathway is characterized by the interaction of the Wnt ligand with its receptor complex, which includes the low-density lipoprotein receptor (LDLR)-related proteins 5/6 and FZD. Ten different FZD genes have been identified in the mammalian genome, encoding seven-pass transmembrane domain structures resembling G-protein-coupled receptors [27].

In the absence of Wnt ligands, the cytoplasmic β-catenin undergoes phosphorylation by an inhibitory complex that includes glycogen synthase kinase 3β (GSK3β), casein kinase I (CK I), Axin, and adenomatous polyposis (APC). This phosphorylation leads to the ubiquitination of β-catenin by E3 polyubiquitin ligase Beta-transducin repeats-containing proteins (β-TrCP) and its subsequent transportation to the proteasome for final degradation. Thus, the lack of Wnt ligands turns off the signals.

When Wnt ligands bind to receptors, an inhibitory complex translocates to the membrane, resulting in phosphorylation of LRP by CK1α and GSK3β, which then recruits disheveled (Dvl) proteins. Dvl inhibits the destructive complex, leading to the accumulation of β-catenin in the nucleus, where it interacts with LEF and TCF proteins in a complex [13]. As a result, transcription factors TCF/LEF are stimulated to initiate transcription of multiple tumor-promoting genes, including c-myc and cyclin D1, resulting in enhanced cell proliferation, invasion, and metastasis. Abnormalities in the expression of different Wnt signaling regulator genes can lead to the upregulation of the pathway and subsequent tumorigenesis [28].

Wnt signaling pathway and non-small cell lung cancer

Wnt signaling plays a crucial role in regulating cellular interactions in the lung. Distinct interactions among epithelial, mesenchymal, immune, and endothelial cells have been recognized in the contexts of development, homeostasis, and disease [29]. The emergence of single-cell RNA sequencing (scRNA-seq) has provided insights into the cell-specific expression patterns of Wnt proteins and receptors within the lung [30]. Increasing evidence shows that the Wnt signaling pathway is a key player in the progression of NSCLC. Components of cigarette smoke that cause LC contribute to the upregulation of Wnt signaling [30]. Malyla et al. reported that treatment with 1% cigarette smoke exposure for 8 days resulted in the upregulation of genes associated with Wnt/β-catenin signaling, including WNT3, DVL3, AXIN1, and β-catenin in 16HBE14o cells, with similar findings observed in human LC patients. However, they noted that the exact mechanism by which cigarette smoke upregulates Wnt signaling requires further investigation [31]. In Wnt-promoted mice, tumors were inhibited when Wnt induction was stopped; however, in p53-mutated mice, tumors became Wnt-independent. β-Catenin was upregulated in 94% of resected squamous cell LC and 51% of adenocarcinomas [32]. Studies have shown that the upregulation of Wnt-1 is clinically associated with poor prognosis. Decreased activity of Wnt signaling by anti-Wnt-1 monoclonal antibody (mAb) or small interfering RNA (siRNA) increased apoptosis in Wnt-1-expressing NSCLC cell lines, reduced NSCLC cell proliferation and xenograft growth, attenuated cell motility and invasion, and promoted a more differentiated phenotype [33].

Lung cancer stem cells (LCSCs) are a small subset of cells with stem-like characteristics in the lung tumor. They exhibit the behavior of embryonic or adult stem cells and are known to initiate and drive metastasis, as well as resistance to anticancer treatments and cancer recurrence, through the constitutive upregulation of developmental signaling pathways, including Wnt signaling [34]. Therefore, targeting the Wnt signaling pathway, which controls LCSC replication, survival, and differentiation, may represent a suitable strategy for cancer therapy [35]. However, to design effective therapeutic approaches, it is essential to explore not only the biological mechanisms of Wnt activation but also the pharmacological interventions that have been developed to inhibit this pathway [36].

Wnt pathway inhibitors

Aberrant activation of the canonical Wnt/β-catenin pathway drives NSCLC progression and therapy resistance. Consequently, multiple strategies to inhibit Wnt signaling are under investigation for LC therapy [35]. Small-molecule inhibitors target Wnt ligand secretion (e.g., the Porcupine inhibitor LGK974, in phase I trials) or downstream complexes. For example, LGK974 has shown potent Wnt inhibition and tumor suppression in lung cancer models, and novel formulations (e.g., cyclodextrin complexes) are being developed to improve its bioavailability and reduce toxicity [37]. Biologics include monoclonal antibodies against Frizzled receptors (e.g., vantictumab/OMP-18R5) or Wnt ligands (e.g., ipafricept/OMP-54F28), as well as ligand traps or decoy receptors. Several of these (e.g., LGK974, OMP-18R5, OMP-54F28, DKN-01) have entered early-phase trials for lung and other cancers [38].

Despite this promise, clinical application of Wnt inhibitors has faced challenges. Broad suppression of Wnt signaling affects normal stem-cell homeostasis, causing dose-limiting toxicity such as gastrointestinal damage and bone loss. Accordingly, current efforts are focusing on targeted delivery and combination therapies [39]. In preclinical NSCLC models, inhibiting the Wnt pathway markedly enhances the efficacy of other agents: for instance, co-inhibiting Wnt components significantly improves EGFR tyrosine kinase inhibitor (TKI) responses in EGFR-mutant LC [40]. Likewise, Wnt inhibition can sensitize tumors to chemotherapy and immune checkpoint blockade by abrogating Wnt-driven drug resistance and immunosuppression [41]. Natural compounds (e.g., flavonoids, terpenoids) and novel delivery systems (liposomes, nanoparticles) are also being explored to achieve more selective Wnt blockade with fewer side effects [42]. In summary, while Wnt pathway inhibitors are scientifically promising therapeutic options for lung cancer, successful clinical translation will require overcoming on-target toxicities through refined targeting, integration with miRNA-based strategies, and rigorous clinical evaluation.

miRNAs: biogenesis and function in lung cancer

MicroRNAs (miRNAs) are diminutive non-coding RNAs, approximately 18–22 nucleotides long, that play a regulatory role by attaching to the 3′-untranslated region (UTR) of their target genes, thereby inhibiting their translation [43]. RNA polymerases II or III are responsible for the transcription of miRNAs, leading to the formation of primary miRNAs (pri-miRNAs). Within the nucleus, RNase III Drosha and its partner DGCR8 cleave pri-miRNAs to generate a hairpin-shaped precursor (pre-miRNA) of about 70 nucleotides [44]. Pre-miRNAs are then exported to the cytoplasm through an Exportin-5/RanGTP-dependent mechanism, where they undergo processing by Dicer to yield a ~ 22 bp double-stranded miRNA. One strand of this duplex is incorporated into the RNA-induced silencing complex (RISC), together with Argonaute (Ago) proteins, TRBP, and PACT, where it guides the complex to complementary mRNA targets to inhibit their translation [44].

The first miRNA, lin-4, was identified in 1993 in Caenorhabditis elegans by the teams of Ambros and Ruvkun. Since then, miRNAs have been shown to modulate the expression of nearly one-third of all human genes, serving as essential regulators of processes such as development, differentiation, apoptosis, and tumorigenesis [45]. Because of this central role, dysregulated miRNAs have been widely studied as potential biomarkers and therapeutic targets in cancer. Indeed, several preclinical studies highlight their therapeutic relevance in NSCLC [46, 47]. For example, miR-124 and miR-142 promote chemosensitivity by targeting SIRT1 to inhibit autophagy, while overexpression of miR-126 enhances radiosensitivity [48]. Furthermore, nanodelivery of tumor-suppressive miRNAs such as let-7b and miR-34a suppresses NSCLC growth and improves survival in animal models [49].

Importantly, recent evidence suggests that alterations in the miRNA biogenesis machinery itself play a critical role in lung carcinogenesis. Dysregulation of Drosha, Dicer, and Exportin-5 disrupts global miRNA maturation and reshapes the oncogenic landscape [50, 51]. Clinical studies show that high Dicer expression is correlated with early-stage NSCLC and better prognosis, whereas elevated Drosha expression, often together with specific miRNAs such as miR-126, is associated with advanced disease and poorer survival [52]. Immunohistochemical analyses further demonstrate that Dicer protein levels increase as NSCLC progresses from stage I to stage III, highlighting the role of altered miRNA processing in tumor evolution [53].

Genetic and epigenetic factors also perturb miRNA maturation in NSCLC. Single-nucleotide polymorphisms within miRNA genes or Drosha/Dicer binding sites can alter expression patterns, while RNA editing events may interfere with pre-miRNA processing [54]. In addition, oncogenic transcription factors such as c-Myc, as well as viral proteins, are capable of modulating Drosha/DGCR8 or Dicer activity, further contributing to abnormal miRNA profiles. These imbalances promote proliferation, epithelial–mesenchymal transition (EMT), metastasis, and resistance to therapy [51]. Therefore, alterations in miRNA processing machinery, particularly dysregulated Drosha and Dicer expression, not only disturb the global miRNA landscape but also directly contribute to NSCLC initiation, progression, and therapeutic resistance [55]. This mechanistic link emphasizes the need to consider biogenesis pathways when designing miRNA-based therapeutic strategies in LC [56].

miRNAs regulating Wnt signaling in lung cancer

Oncogenic miRNAs

Oncogenic miRNAs, referred to as oncomiRs, are acknowledged for their role in suppressing the translation of tumor suppressor genes and are found to be upregulated in cancer. When overexpressed, these miRNAs confer various properties to tumors including enhanced proliferation, epithelial-mesenchymal transition (EMT), metsastasis, and attenuated apoptosis [57]. In this section, we will review several miRNAs that are upregulated in LC and are known to promote the Wnt pathway and LC progression (Fig. 1).

Fig. 1.

The summary of oncogenic microRNAs (miRNAs) regulating Wnt signaling pathway in lung cancer. Represented miRNAs that are highly expressed in LC bind to specific target gene encoding proteins involved in Wnt signaling pathway deactivation and repress their expression. This event causes upregulation of Wnt signaling and the appearance of tumor characteristics. CKI Casein kinase I, FAF 1 Fas-associated factor 1, SFRP secreted frizzled-related protein, Dvl Dishevelled, Dkk Dickkopf, GSK-3 beta Glycogen synthase kinase-3 beta, TCF/LEF T-cell factor/lymphoid enhancer factor

Fan et al. demonstrated that simultaneous upregulation of the miR-23a/27a/24-2 cluster is associated with enhanced Wnt signaling through targeting its inhibitors and promoting methylation-induced silencing of tumor suppressor genes. This cluster promotes sphere formation, soft agar colony growth, and metastasis in large-cell lung carcinoma and adenocarcinoma cell lines. Consequently, the simultaneous upregulation of these three miRNAs is considered a reliable biomarker for predicting postoperative recurrence and prognosis in early-stage NSCLC, and their inhibition may represent a promising therapeutic approach [58].

Another example is miR-19, which targets and downregulates p38α kinase, thereby overactivating Wnt signaling by repressing pathway inhibitors and stimulating pathway enhancers. LRP6, one of the overexpressed components of Wnt signaling in this context, interacts with R-spondin receptors such as Lgr6. Cells with high Lgr6 expression can respond robustly to Wnt activation, enhancing tumor growth and progression, particularly in late-stage NSCLC. Thus, miR-19 has been suggested as a prognostic, diagnostic, and therapeutic target for NSCLC [59] (Table 1).

Table 1.

Preclinical Potentials of miRNAs in regulating Wnt pathway and lung cancer models

Type miRNA	Wnt effect	Tumor effects	Study	Reference
miR-34a	Directly targets Wnt1 and β-catenin, reducing Wnt signaling activity	Inhibits LC cell proliferation and invasion; enhances the efficacy of cisplatin in LC cells	Mouse models	[60, 61]
miR-200 family (miR-200a, miR-200b, miR-200c)	Targets ZEB1/ZEB2 and indirectly suppresses Wnt/β-catenin signaling	Reduces EMT, metastasis, and tumor growth	Lung cancer models	[62–64]
miR-145	Targets Wnt3a and β-catenin, suppressing Wnt signaling	Inhibits EMT and reduces LC cell migration and invasion	Lung cancer models	[65, 66]
miR-375	Downregulates Wnt1 and β-catenin expression	Suppresses LC cell migration and invasion	In vitro	[67, 68]
miR-21	Regulates Wnt/β-catenin signaling	Promotes chemoresistance; inhibition of miR-21 enhances cisplatin sensitivity; correlates with poor prognosis	In vitro/lung cancer models	[69–71]
miR-221	Targets Wnt signaling components to modulate drug resistance	Downregulation of miR-221 sensitizes LC cells to paclitaxel	Lung cancer models	[72–74]
miR-128	Targets β-catenin	Reduces self-renewal and tumor-initiating capacity of lung CSCs	In vitro	[75, 76]
miR-129	Downregulates Wnt5a and β-catenin	Suppresses lung CSC properties and tumor growth	Lung cancer models	[77, 78]
miR-126	Inhibits Wnt/β-catenin signaling, reducing VEGF expression	Suppresses tumor angiogenesis and growth	Lung cancer models	[79, 80]

miR-21 as a central oncomiR in NSCLC

miR-21 is among the most commonly upregulated microRNAs in malignancies and plays a central role in modulating cell growth, apoptosis, motility, and metastasis. It has been observed to be significantly overexpressed in LC tissues compared to adjacent non-cancerous tissues [73]. Elevated miR-21 levels correlate with enhanced proliferation and reduced apoptosis, partly through altered expression of Bax, Bcl-2, cyclin D1, and cyclin E1. In LC cells treated with a miR-21 inhibitor, phosphorylated β-catenin levels are significantly reduced, while total β-catenin remains unaffected [73, 74].

This phenomenon can be mechanistically explained by the ability of miR-21 to repress tumor suppressors such as PTEN, thereby activating the PI3K/AKT pathway [75]. Activated AKT inhibits GSK3β, which reduces β-catenin phosphorylation and promotes its nuclear accumulation without altering total β-catenin protein levels. In addition, activation of the EGFR/RAS pathway induces miR-21 transcription, establishing a positive feedback loop that reinforces Wnt/β-catenin signaling and contributes to resistance against EGFR-TKIs and chemotherapy [76]. Conversely, competing endogenous RNAs such as lncRNA GAS5 can sponge miR-21 and mitigate its oncogenic effects. Collectively, these findings clarify the regulatory link between miR-21 and phosphorylated β-catenin, highlighting its dual role in Wnt pathway activation and therapy resistance [77]. Nobiletin, a natural compound with anticancer properties, has been shown to inhibit NSCLC progression by downregulating miR-15-5p, which normally targets Wnt pathway inhibitors such as NKD1, WIF1, and AXIN2 [78]. Similarly, MYPT1, a negative regulator of Wnt signaling, is suppressed by miR-19b-3p in lung adenocarcinoma tissues, thereby activating Wnt signaling and promoting oncogenesis [79, 80] (Fig. 2).

Fig. 2.

Regulation of Wnt/β-catenin signaling by miRNAs can be summarized as follows: On the left, miRNAs influence inactive Wnt/β-catenin signaling to promote epithelial-to-mesenchymal transition (EMT). In the absence of Wnt ligands, β-catenin is phosphorylated by GSK3β within a destruction complex that includes Axin, APC, CKIα, and GSK3β, leading to β-catenin degradation via ubiquitination. Here, miRNAs support EMT by targeting suppressors of Wnt/β-catenin signaling. On the right, miRNAs act on activated Wnt/β-catenin signaling to inhibit EMT. When Wnt ligands bind to receptors, the phosphorylation of β-catenin by GSK3β is blocked, allowing β-catenin to detach from the destruction complex and accumulate in the cytoplasm. Subsequently, β-catenin moves into the nucleus, where it partners with TCF/LEF to activate transcription of Wnt target genes like Twist and Snail, promoting EMT. In this context, miRNAs hinder EMT by targeting various elements of the Wnt/β-catenin signaling pathway

Additional oncogenic miRNAs in NSCLC

AXIN1, a component of the β-catenin destruction complex, is inhibited by miR-55, which enhances malignant behavior in NSCLC. SFRPs, which normally sequester Wnt ligands extracellularly, are targeted by miR-106b-5p, miR-27a, and miR-1260b, all of which accelerate NSCLC progression [81, 82]. FAF1, a promoter of β-catenin ubiquitination and degradation, is silenced by miR-26a-5p, thereby contributing to Wnt/β-catenin upregulation and tumorigenesis [83].

Overexpression of miR-708 has been linked to poor survival in non-smoker lung adenocarcinoma patients by targeting TMEM88, a negative regulator of Wnt signaling [84]. Similarly, miR-582-3p downregulates multiple Wnt inhibitors (AXIN2, DKK3, SFRP1), enhancing CSC-like properties and promoting tumor recurrence. MiR-650 is also upregulated in NSCLC and contributes to proliferation and invasion by activating the ING4/Wnt1/β-catenin signaling pathway [85, 86]. Overall, these oncogenic miRNAs drive tumor initiation and progression by stimulating cell cycle activity, proliferation, and EMT while suppressing apoptosis. Designing drugs that specifically inhibit these oncomiRs may represent a promising therapeutic strategy for NSCLC [87, 88].

Tumor suppressor miRNAs

Tumor suppressor miRNAs could inhibit proliferation, metastasis, migration, and eventually tumor development by silencing the expression of tumor stimulator genes or oncogenes. Tumor suppressor miRNAs are downregulated in cancers [89–92]. Rescuing the expression of these miRNAs could reverse the tumor phenotype [92]. Here, we summarize tumor suppressor miRNAs that are reduced in lung cancer (LC) and negatively modulate the Wnt pathway, thereby suppressing LC progression (Fig. 3).

Fig. 3.

Summary of suppressor miRNAs regulating Wnt signaling pathway in lung cancer. Illustrated miRNAs that are lowly expressed in lung cancer bind to specific target gene encoding proteins involved in the promotion of Wnt signaling pathway and repress their expression. This event prevents the upregulation of Wnt signaling and the appearance of tumor characteristics. CKI Casein kinase I, SFRP secreted frizzled-related protein, Dvl Dishevelled, Dkk Dickkopf, GSK-3 beta Glycogen synthase kinase-3 beta, TCF/LEF T-cell factor/lymphoid enhancer factor

circ_0017109–miR-671-5p–FZD4 axis: circ_0017109 functions as an oncogenic circRNA in NSCLC by sponging miR-671-5p in A549 and H1299 cells. miR-671-5p targets FZD4 and inhibits its translation. Thus, circ_0017109 increases FZD4 levels and promotes NSCLC cell proliferation and survival through Wnt activation [93].

miR-1253–NOVA2–β-catenin stability: NOVA2 has been identified as a β-catenin RNA-binding protein that enhances β-catenin mRNA stability. Reduced miR-1253 leads to NOVA2 upregulation and Wnt pathway activation, promoting LC progression [94].

miR-29a-3p and miR-107 against Wnt3a/β-catenin: miR-29a-3p is reduced in NSCLC cell lines and in a lung adenocarcinoma mouse model. Its overexpression decreases proliferation, migration, invasion, tumor growth, and reduces Wnt3a and β-catenin protein levels. miR-107 similarly downregulates Wnt3a and attenuates proliferation, migration, and EMT in lung adenocarcinoma [95, 96].

circVAPA–miR-876-5p–WNT5A: circVAPA is upregulated in NSCLC. Its knockdown suppresses growth, invasion, stemness, and tumor growth in mice. Mechanistically, circVAPA sponges miR-876-5p to increase WNT5A, thereby stimulating Wnt/β-catenin signaling; the effects are reversed by miR-876-5p restoration or WNT5A overexpression [97, 98].

miR-326 and miR-577 against Wnt2b: Both are downregulated in NSCLC; restoring them inhibits Wnt2b and attenuates malignant phenotypes. miR-29c also suppresses Wnt signaling and LC development, supporting its therapeutic restoration [99, 100].

miR-590–YAP and Wnt output: Yes-associated protein 1 (YAP1) can act as a positive regulator of Wnt output. miR-590 inhibits YAP1, suppressing Wnt signaling and NSCLC progression, suggesting prognostic utility for miR-590 [101]. Consistently, miR-532-3p targets FOXR2 and reduces β-catenin, cyclin D1, and c-Myc, thereby blocking NSCLC progression. miR-4429 and miR-216a also target β-catenin to inhibit lung adenocarcinoma growth [102].

Clarifying the YES1–β-catenin link implicated by the circ-ZNF124–miR-498 axis

What the original data showed: circ-ZNF124 is upregulated in NSCLC. Its knockdown reduces proliferation and invasion, increases apoptosis, causes cell-cycle arrest, and limits tumor growth in vivo [103]. circ-ZNF124 sponges miR-498, which directly targets YES1. Downregulating circ-ZNF124 elevates miR-498, decreases YES1, and inactivates β-catenin and c-Myc expression. The missing piece was how YES1 connects to β-catenin activity [104].

Mechanistic bridge: Hippo/YAP–Wnt crosstalk controlled by YES1: The Src-family kinase YES1 phosphorylates YAP1 at sites such as Y357, stabilizing and activating YAP1. Activated YAP1 cooperates with β-catenin in the nucleus; YAP1 can form complexes with β-catenin and TBX5 to drive transcription of proliferation and survival genes [105]. In multiple models, including lung cancer, inhibiting YES1 or YAP1 disrupts this nuclear complex and reduces β-catenin-dependent transcriptional output. Agents like dasatinib that inhibit YES1/Src decrease nuclear YAP1 and β-catenin activity and induce apoptosis in EGFR-mutant LC cells. Thus, EGFR signaling can signal through YES1 to increase nuclear YAP1, which then amplifies β-catenin oncogenic transcription [105, 106].

Putting it together for circ-ZNF124–miR-498: When circ-ZNF124 is abundant, it sponges miR-498, relieving repression of YES1. Elevated YES1 enhances YAP1 activation, which partners with β-catenin in the nucleus to boost Wnt target gene expression. When circ-ZNF124 is downregulated, miR-498 increases, YES1 falls, YAP1 activation declines, and the cooperative YAP1–β-catenin transcriptional program is blunted [107].

Additional suppressor circuits: miR-485-5p–WLS: miR-485-5p targets WLS, reducing Wnt ligand secretion and inactivating Wnt signaling; in lung adenocarcinoma, SNHG17 sponges miR-485-5p, restoring WLS and promoting growth and migration [108].

miR-489-3p–USP48–β-catenin: miR-489-3p is decreased in NSCLC. It targets USP48, a deubiquitinase that stabilizes β-catenin. Restoring miR-489-3p increases β-catenin ubiquitination, reduces Wnt signaling, and limits growth and metastasis in vivo [108, 109]. miR-1182–KLF8. KLF8 promotes β-catenin accumulation and oncogenic transformation. miR-1182 binds KLF8 mRNA and suppresses its translation; low miR-1182 in NSCLC facilitates tumor progression [110].

miR-149-3p–MAZ: Upregulation of miR-149-3p by the Jin formula reduces proliferation, migration, invasion, and tumor growth by targeting MAZ and downregulating Wnt/β-catenin signaling in models of LC [111, 112]. Across these examples, tumor suppressor miRNAs converge on β-catenin availability, nuclear co-activator function, ligand secretion, and destruction-complex integrity. Their restoration can dampen Wnt output and restrain NSCLC progression [113] (Table 2).

Table 2.

Tumor-suppressive miRNAs regulating Wnt signaling in lung cancer

miRNA type	Wnt-related targets/effect	Tumor effects in lung cancer	Study type/model	Reference
miR-671-5p	Targets FZD4; blocks Wnt receptor translation	Inhibits proliferation and survival; circ_0017109 sponges miR-671-5p to reactivate Wnt signaling	In vitro (A549, H1299)/NSCLC models	[93]
miR-1253	Suppresses NOVA2, reducing β-catenin mRNA stability	Downregulation activates Wnt signaling and promotes LC progression	In vitro/NSCLC tissues	[94]
miR-29a-3p, miR-107	Target Wnt3a and β-catenin; reduce Wnt signaling output	Decrease proliferation, migration, invasion, and tumor growth	In vitro/mouse lung adenocarcinoma models	[95, 96]
miR-876-5p	Targets WNT5A; circVAPA sponges miR-876-5p	Overexpression suppresses growth, invasion, and stemness; circVAPA knockdown reverses effects	In vitro/mice	[97, 98]
miR-326, miR-577, miR-29c	Target Wnt2b and other Wnt activators	Inhibit malignant phenotypes and support therapeutic restoration strategies	NSCLC cell lines/animal models	[99, 100]
miR-590	Targets YAP1, reducing Wnt transcriptional output	Suppresses proliferation and tumor growth; prognostic potential in NSCLC	In vitro/NSCLC tissues	[101]
miR-532-3p, miR-4429, miR-216a	Target FOXR2 or β-catenin directly	Decrease cyclin D1 and c-Myc, blocking proliferation and EMT	In vitro/lung adenocarcinoma	[102]
miR-498	Targets YES1, reducing YAP/β-catenin nuclear co-activation	Loss of circ-ZNF124 increases miR-498, suppressing YES1 and tumor growth	In vitro/in vivo NSCLC	[103–107]
miR-485-5p	Targets WLS, blocking Wnt ligand secretion	Reduces migration and invasion; SNHG17 sponges miR-485-5p to restore Wnt output	In vitro/adenocarcinoma	[108]
miR-489-3p	Targets USP48, promoting β-catenin ubiquitination and degradation	Inhibits growth and metastasis in vivo	In vitro + xenograft models	[108, 109]
miR-1182	Targets KLF8, reducing β-catenin accumulation	Suppresses proliferation and transformation in NSCLC	In vitro	[110]
miR-149-3p	Targets MAZ, inhibiting Wnt/β-catenin activation	Reduces proliferation, migration, invasion, and tumor growth	In vitro/animal models	[111, 112]

miRNAs and other ncRNAs in drug resistance of lung cancer

In recent decades, chemotherapeutic drugs have improved cancer outcomes, but drug resistance remains a major obstacle, particularly for patients with advanced NSCLC [114]. miRNAs can modulate drug response by regulating key pathways such as Wnt/β-catenin, PI3K/AKT, and Hippo signaling. Consequently, modulating these miRNAs and related noncoding RNAs (ncRNAs) offers promising avenues to overcome chemoresistance [115, 116].

Cisplatin resistance: Zhang et al. found upregulation of the oncogenic miR-181c in NSCLC tissues and cisplatin-resistant cells, where it represses WIF1 and activates Wnt signaling, promoting resistance. Similarly, miR-155 is frequently overexpressed in NSCLC and directly promotes cisplatin resistance by blocking apoptosis through TP53-associated feedback loops [117]. High miR-155 levels correlate with poor prognosis, and its inhibition sensitizes tumors to platinum drugs, though immune effects via PD-L1 regulation must be considered [118]. Conversely, tumor suppressor miRNAs such as miR-140-3p increase cisplatin sensitivity and reduce cancer stemness by inhibiting β-catenin/TCF activity. The lncRNA SNHG1 also contributes to cisplatin resistance by sponging miR-140-5p, thereby upregulating Wnt signaling in A549 and H1299 cells [119].

Taxane and paclitaxel resistance: Oncogenic miR-1260b represses SFRP1 and confers taxane resistance in NSCLC. Additionally, miR-221 has been implicated in paclitaxel resistance through Wnt and PI3K pathway activation, where its inhibition restores sensitivity to treatment [120].

Gemcitabine and other agents: Recent studies highlight the role of ncRNAs in resistance to gemcitabine. For example, the lncRNA MALAT1 is overexpressed in gemcitabine- and cisplatin-resistant cells, where it sponges miR-145 and promotes KLF4 expression, thereby driving survival signaling [121]. Knockdown of MALAT1 restores chemosensitivity in resistant LC cells. Circular RNAs have also emerged as modulators of gemcitabine resistance; circRNAs that sequester miR-398 or miR-498 derepress oncogenic mediators in Wnt/PI3K signaling, promoting resistance [122].

To overcome resistance, antagonists of oncogenic miRNAs (e.g., antagomirs against miR-181c or miR-155) and restoration of tumor-suppressive miRNAs (e.g., miR-140-3p) represent viable options. Delivery systems such as nanoparticles carrying anti-miR-155 oligonucleotides or miR-140 mimics have shown promise in preclinical studies [123, 124]. Combinatorial approaches, such as pairing chemotherapy with YAP inhibitors or ncRNA modulators, may provide synergistic benefits by breaking chemoresistance circuits [125]. Beyond chemoresistance, aberrant activation of the Wnt/β-catenin pathway also contributes to immune evasion in NSCLC. Wnt-driven tumor cells secrete immunosuppressive cytokines such as CCL4 and IL-10, which induce M2 macrophage polarization and inhibit CD4⁺ and CD8⁺ T-cell infiltration (Fig. 4). Moreover, Wnt activation promotes endothelial remodeling and the formation of tumor-like vascular networks, creating a physical and metabolic barrier that weakens anti-tumor immunity. Therefore, combining Wnt-targeted miRNA therapeutics with immunomodulatory agents, such as PD-1/PD-L1 inhibitors or macrophage reprogramming strategies, may simultaneously overcome drug resistance and restore immune responsiveness in advanced NSCLC [122–126].

Fig. 4.

Wnt signaling plays a crucial role in modulating various aspects of the tumor microenvironment. When activated in cancer cells, Wnt signaling can stimulate the proliferation of endothelial cells and the formation of capillary-like networks by influencing angiogenic factors. Additionally, reduced levels of chemokines such as CCL4 and IL-10 hinder the recruitment of dendritic cells. The endogenous activation of β-catenin in tumor cells may lead to resistance against effector and memory T cells, while also affecting the activation of liver NKT cells. Hepatic tumor cells promote M2 macrophage polarization through the activation of the canonical Wnt/β-catenin signaling pathway. Furthermore, Wnt ligands released by macrophages can activate Wnt signaling in cancer cells, enhancing malignancy, and in hepatic progenitor cells, facilitating their differentiation into hepatocytes. This signaling can also contribute to protumoral activation and the self-renewal of cancer stem cells

Therapeutic opportunities of microRNAs for LC

Building on the preclinical evidence outlined in earlier sections, therapeutic strategies have been developed to directly modulate miRNA activity in NSCLC [126]. These approaches mainly include two complementary directions: inhibition of oncogenic miRNAs and restoration of tumor suppressor miRNAs. For the first strategy, antagomirs, locked nucleic acids (LNAs), sponges, and CRISPR/Cas9-based tools have been designed to specifically silence overexpressed miRNAs that drive tumor progression [126, 127]. For the second, synthetic mimics are applied to replenish downregulated tumor suppressor miRNAs, delivered through viral vectors (lentiviral, adenoviral, retroviral) or non-viral platforms such as lipid nanoparticles (LNPs) and exosomes. These interventions aim to suppress proliferation, invasion, and drug resistance while restoring apoptosis and chemosensitivity [128, 129].

Several clinical trials have attempted to translate these concepts into practice. For example, MesomiR-1 (a miR-16 mimic delivered via a lentiviral vector) demonstrated safety and stable disease in some NSCLC patients [130]. MRX34, a liposomal formulation of miR-34a, showed promising preclinical activity but was discontinued due to immune-related toxicities [131]. More recently, the ongoing INT-1B3 trial, which employs LNP-loaded miR-193a-3p mimics, has reported encouraging preclinical data by simultaneously downregulating multiple oncogenic pathways [132]. Together, these studies highlight both the feasibility and the challenges of moving miRNA-based therapies from bench to bedside.

Limitations of clinical trials

Despite these advances, early clinical studies of miRNA-based therapies have revealed important limitations [133]. For example, the MesomiR-1 trial produced only transient cytokine release without durable tumor responses, while the MRX34 trial, which tested a liposomal miR-34a mimic, was halted due to severe immune-related toxicities including cytokine storms and fatal adverse events [131, 134]. These outcomes underscore recurring challenges: systemic miRNA delivery can activate innate immunity, organ sequestration (particularly hepatic accumulation) reduces effective bioavailability, and synthetic carriers may induce off-target inflammation [135]. Moreover, most early trials have been limited by small, heterogeneous, heavily pretreated patient populations, restricting the ability to detect robust efficacy signals [136].

Translational barriers to miRNA therapeutics

Translating miRNA–Wnt findings into NSCLC treatments faces multiple hurdles. Efficient and specific delivery remains a primary obstacle: miRNAs are inherently unstable and negatively charged, requiring carriers (lipid nanoparticles, viral or exosomal vectors) that must achieve adequate tumor penetration without provoking toxicity [137]. Importantly, each miRNA has many potential mRNA targets, so off-target gene regulation is a serious concern [138].

Chemical modifications and nanoparticle excipients used to stabilize miRNAs can themselves activate immune sensors such as Toll-like receptors or complement pathways, leading to inflammation or hypersensitivity [138]. Moreover, tumor heterogeneity in NSCLC (genetic and microenvironmental diversity) means that a single miRNA therapy may be effective in only a subset of cells or patients [139]. Finally, regulatory and manufacturing complexity is significant: agencies require rigorous proof of safety, consistent large-scale production, and comprehensive pharmacokinetic data for these advanced therapeutics [140]. Overlapping patents on miRNA sequences and delivery technologies, along with the need for scalable GMP manufacturing, further complicate clinical translation (Table 3).

Table 3.

Therapeutic opportunities and clinical applications of microRNAs in lung cancer

Therapeutic strategy	Mechanism/target	Delivery platform/model	Clinical or preclinical outcome	Reference
Antagomirs/LNAs	Inhibit oncogenic miRNAs such as miR-21, miR-155, and miR-181c to suppress Wnt, PI3K, and apoptotic escape pathways	Chemically modified oligonucleotides (antagomirs, LNAs); administered systemically or via nanoparticles	Reduced proliferation and chemoresistance; re-sensitization to cisplatin and paclitaxel in NSCLC models	[126, 127]
miRNA mimics (tumor-suppressor restoration)	Restore downregulated miRNAs such as miR-34a, miR-16, and miR-193a-3p to repress oncogenic signaling	Synthetic mimics delivered through viral vectors (lentiviral, adenoviral) or lipid nanoparticles (LNPs)	Enhanced apoptosis, decreased tumor growth, and inhibition of β-catenin and PI3K/AKT activation	[128, 129]
Exosome-based miRNA delivery	Utilize tumor-targeted exosomes to encapsulate therapeutic miRNA mimics or inhibitors	Engineered exosomes derived from MSCs or immune cells	Improved stability, reduced immune activation, and efficient tumor penetration in NSCLC models	[128, 130]
MesomiR-1 (miR-16 mimic)	Reconstitutes miR-16 levels to suppress multiple oncogenic pathways (Wnt, Bcl-2, CCND1)	Lentiviral vector formulation tested in phase I trial	Demonstrated safety and partial disease stabilization in some NSCLC patients	[130]
MRX34 (miR-34a mimic)	Targets β-catenin, c-Myc, and PD-L1 pathways to restore apoptosis and immune surveillance	Liposomal formulation; phase I clinical trial	Preclinical efficacy observed; trial halted due to immune-related toxicities (cytokine storms)	[131, 134]
INT-1B3 (miR-193a-3p mimic)	Simultaneously downregulates Wnt, MAPK, and PI3K pathways	LNP-based systemic delivery (ongoing trial)	Encouraging preclinical results; early-phase data suggest multi-pathway inhibition with manageable safety	[132]
Combination therapies	Pair miRNA therapeutics with chemotherapy, YAP inhibitors, or immunotherapies	Nanoparticle-mediated co-delivery systems or sequential regimens	Show synergistic tumor suppression and reversal of drug resistance and immune evasion	[126–128]

Benefits, drawbacks, and standardization efforts in employing miRNAs as LC biomarkers

LC patients often face numerous challenges as their disease progresses, leading to unfavorable clinical outcomes, complications, and recurrence [141]. Timely detection of LC is crucial for effective disease management and prevention of advancement. However, the absence of sensitive and practical early diagnostic techniques remains a significant barrier [142]. Although tumor biopsy is considered the definitive diagnostic method, it is invasive and impractical for longitudinal monitoring. Moreover, biopsy represents only a single time point of tumor biology [143].

Therefore, alternative biomarkers that can be repeatedly assessed are needed. Liquid biopsy has emerged as a promising approach for detecting circulating tumor biomarkers and offers a minimally invasive means of capturing tumor dynamics [144]. Liquid biopsy analytes include cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), microRNAs (miRNAs), exosomes, and circulating tumor cells (CTCs) [145]. miRNAs are particularly attractive as they play crucial roles in LC initiation, progression, and metastasis as oncogenes, tumor suppressors, and signaling regulators. Exosomal miRNAs, which are secreted into body fluids such as plasma, serum, pleural effusions, saliva, and cerebrospinal fluid, have demonstrated potential as stable and effective non-invasive biomarkers for early diagnosis, tumor profiling, and treatment monitoring [146]. Quantification methods include qPCR (e.g., TaqMan), next-generation sequencing (NGS), and hybridization-based assays [147].

Despite this promise, limitations remain. Many circulating miRNAs are not secreted in sufficient quantities, some are unstable and prone to degradation, and inconsistent isolation methods have hindered reproducibility. Most importantly, there is no universal standardized protocol for pre-analytical processing, normalization, and data interpretation. This lack of standardization has contributed to inconsistent biomarker signatures and limited clinical translation [148, 149].

Ongoing efforts to address these challenges: Recent initiatives are actively working toward assay standardization and methodological harmonization. The MIQE (Minimum Information for Quantitative PCR Experiments) guidelines have been extended to miRNA assays, requiring detailed reporting of RNA quality, reverse transcription efficiency, and the use of validated reference genes [150]. Large collaborative projects, such as the Extracellular RNA Communication Consortium, are establishing best practices for exosomal miRNA isolation and quantitation, including droplet digital PCR and locked nucleic acid probes [151, 152]. Moreover, multicenter validation studies are underway, where matched protocols are applied across different cohorts to identify robust and reproducible LC signatures [153]. Novel analytical platforms, including digital microfluidics and RNA sequencing with synthetic spike-ins, are also being tested for improved accuracy and dynamic range [154]. While circulating miRNAs hold strong potential as non-invasive biomarkers, their clinical utility depends on overcoming technical barriers. Ongoing collaborative standardization efforts are expected to improve reproducibility and accelerate the translation of miRNA assays into routine LC screening and monitoring [155, 156].

Conclusion

The intricate relationship between microRNAs and the Wnt signaling plays a pivotal role in the pathogenesis of LC, presenting both biological implications and significant clinical potential. miRNAs regulate Wnt signaling by targeting genes involved in the pathway. We have summarized a series of interactions between miRNAs and the gene transcripts involved in the canonical Wnt signaling cascade in LC. Suppression of oncogenic miRNAs or replenishment of tumor suppressor miRNAs could reverse the cancer phenotype. In addition, quantification of miRNA expression may be used for tumor diagnosis and prognosis. A more comprehensive discovery of miRNAs and the Wnt signaling regulatory network will provide useful insights to further the development of miRNA-based therapies.

Although microRNAs represent promising therapeutic tools for targeting the Wnt signaling pathway, their translation into clinical applications remains challenging. The main limitations include their low stability in biological fluids, difficulties in achieving efficient and tissue-specific delivery, and the possibility of unintended interactions with non-target genes. In addition, the complex regulatory networks governed by microRNAs may generate unpredictable downstream effects. Recent investigations are focusing on advanced delivery strategies such as lipid-based nanoparticles, exosome carriers, and viral vector systems to improve stability, targeting precision, and therapeutic outcomes. Overcoming these barriers is essential for moving microRNA-based interventions from experimental models toward clinical implementation in lung cancer treatment.

While microRNA based interventions against the Wnt pathway remain compelling, their successful clinical translation will depend on resolving delivery barriers, minimizing immune and off target effects, and demonstrating consistent target engagement in tumor tissue. Integrating biomarker driven selection, optimized lung directed delivery, and mechanism aligned endpoints into prospective trials should accelerate the path from proof of concept to durable clinical benefit.

Abbreviations

APC

Adenomatous polyposis coli

β-catenin

Beta-catenin

CK1

Casein kinase 1

CSC

Cancer stem cell

ctDNA

Circulating tumor DNA

DVL (Dvl)

Dishevelled

EGFR

Epidermal growth factor receptor

EMT

Epithelial–mesenchymal transition

FZD

Frizzled receptor

GSK3β

Glycogen synthase kinase 3 beta

LC

Lung cancer

LCSCs

Lung cancer stem cells

LEF

Lymphoid enhancer factor

lncRNA

Long non-coding RNA

LRP5/6

Low-density lipoprotein receptor-related protein 5/6

miRNA

MicroRNA

NGS

Next-generation sequencing

NSCLC

Non-small cell lung cancer

PTEN

Phosphatase and tensin homolog

qPCR

Quantitative polymerase chain reaction

RISC

RNA-induced silencing complex

SFRP

Secreted frizzled-related protein

TCF

T-cell factor

TME

Tumor microenvironment

VEGF

Vascular endothelial growth factor

WIF1

Wnt inhibitory factor 1

WLS

Wnt ligand secretion mediator

Wnt

Wingless-type integration site

LC

Long Cancer

Author contributions

Yahui Wang 1, Saodat Yarmukhamedova 2, Rawaa Najim Alkhamessi 3, Mustafa Jawad Kadham 4, Raed Fanoukh Aboqader 5,Vishal Thakur 6, Rana hussein naser 7, Natrayan Lakshmaiya 8, PREMKUMAR J 9, Wesam Taher Almagharbeh 10, Ioan Sârbu 11*, Fitra Ari Aditya 12, Carmen Iulia Ciongradi 13, sina hamzehzadeh: Conceptualization, Writing—original draft, Writing—review & editing.

Funding

Self-funded by the authors.

Data availability

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.