2.1 MicroRNAs

Small ncRNAs, characterized by their length of 20–30 nucleotides and their association with Argonaute family proteins, are categorized into three classes in animals: miRNAs, siRNAs, and PIWI-interacting RNAs [37]. Among these, miRNAs are the predominant class of small RNAs in most somatic tissues. Over 60% of human protein-coding genes have at least one conserved miRNA-binding site. When including the numerous non-conserved sites, it is likely that most protein-coding genes are regulated by miRNAs. miRNAs are small RNAs (approximately 22 nucleotides) that regulate gene expression post-transcriptionally by binding to the 3' untranslated regions (UTR) of target mRNAs, leading to their degradation or translational repression [37]. The biogenesis of miRNAs begins in the nucleus, where primary miRNAs are transcribed by RNA polymerase II, then processed by the Drosha-DGCR8 complex into precursor miRNAs, which are then exported to the cytoplasm. In the cytoplasm, Dicer further processes precursor miRNAs into mature miRNAs, which are incorporated into the RNA-induced silencing complex (RISC) to regulate target mRNAs [38,39].

The most recent version of the miRBase database (v22) includes 38,589 entries representing hairpin precursor microRNAs from 271 organisms, though the functional relevance of many remains to be elucidated [40]. The dysregulation of miRNAs has been associated with cancer development and progression in various cancers [38]. For example, in pancreatic cancer with mutant KRAS, RAS-responsive element-binding protein 1 (RREB1) represses the promoters of miR-143 and miR-145. Simultaneously, both KRAS and RREB1 are targets of miR-143 and miR-145, creating a feedforward mechanism that amplifies the effect of RAS signaling [41]. The clinical utility of miRNAs is highlighted by aberrant miRNA levels indicating the physiological state of cancer cells, which can be detected through miRNA expression profiling and utilized for diagnosis and prognosis. In fact, miRNA profiling is often more accurate than mRNA profiling for classifying tumors because miRNA expression closely correlates with tumor origin and stage. It can also effectively classify poorly differentiated tumors, which are challenging to identify using standard histological methods [42–45]. Furthermore, circulating miRNAs in serum may serve as diagnostic tools. For instance, miR-141 expression levels in serum were reported to be elevated in patients with prostate cancer compared to healthy control individuals [46,47].

2.2 Long non-coding RNAs

Over the past decade, research has increasingly shown that the genomes of various species are extensively transcribed, leading to the production of numerous lncRNAs. lncRNAs are longer than 200 nucleotides and participate in a wide range of regulatory functions, including chromatin remodeling, transcriptional regulation, and post-transcriptional processing [48–50]. This broad definition includes a vast and highly diverse collection of intergenic transcripts, eRNAs, and sense or antisense transcripts overlapping other genes that vary in their biogenesis and genomic origin [51]. Most lncRNAs are transcribed by RNA polymerase II and undergo similar processing steps as mRNAs, including capping, splicing, and polyadenylation. However, lncRNAs typically do not encode proteins. Instead, they function through various mechanisms, including acting as molecular scaffolds, guides, decoys, and enhancers [49]. The resulting lncRNAs are typically capped with 7-methylguanosine at their 5′ ends and polyadenylated at their 3′ ends [52].

Data from the Human GENCODE project indicate that the human genome contains over 16,000 lncRNA genes; however, other estimates suggest there could be more than 100,000 human lncRNAs [53,54]. These lncRNAs are implicated in acquiring all the hallmarks of cancer, ranging from intrinsic abilities like enhanced proliferation and survival to changes in metabolism and interactions with the tumor microenvironment (TME). They are transcriptionally regulated by key oncogenic or tumor-suppressive transcription factors, including p53 [55,56], MYC [57,58], the estrogen receptor [59], and signaling pathways such as Notch [60]. This regulation allows lncRNAs to contribute significantly to oncogenic or tumor-suppressive responses. Their tissue- and condition-specific expression patterns suggest strong potential as biomarkers as well, making them promising targets for clinical interventions. One such lncRNA, the tissue differentiation-inducing non-protein coding RNA (TINCR), is notably dysregulated in various cancers and influences tumor development and progression [61].

2.3 Circular RNAs

circRNAs are produced through an inefficient back-splicing process, in which a downstream splice donor is joined to an upstream splice acceptor, thus creating a covalently closed looped structure that lacks poly-adenylated tails. This process largely utilizes the canonical RNA splicing machinery. Most current research has focused on circRNAs derived from known genes, where in some cases, the circular isoform is much more predominant than its linear counterpart [10]. While escaping debranching can lead to the formation of intronic circRNAs, different mechanisms are involved in the generation of circRNAs comprising exonic sequences [62]. Various regulatory sequences [63,64] and protein factors [65–68] are implicated in this process, with circRNAs being produced both co-transcriptionally and post-transcriptionally [69,70]. circRNAs can act as miRNA sponges, regulate transcription, and interact with RNA-binding proteins.

The number of unique circRNAs produced in human cells (approximately 100,000) [71] is almost fivefold higher than that of protein-coding genes (approximately 20,000) [71–74]. Despite their widespread presence, most circRNAs have not been functionally characterized, and their biological roles remain largely unknown. However, their clinical utility is highlighted through their association with various physiological conditions and cellular features as well as with clinical characteristics such as histological grade, tumor size, metastasis stage, and cancer aggressiveness [75,76]. For example, CiRS-7/CDR1as is highly abundant in intratumoral stromal cells, which are widely used as a prognostic factor in colon and breast carcinomas [77,78]. Furthermore, the tissue and cell-type specificity enables better correlation with specific pathologies, allowing for improved differentiation between cancer subtypes [79–82].

2.4 Transfer RNAs

tRNAs are small ncRNA molecules ranging from 56 to 80 nucleotides long and represent a unique clover-leaf secondary structure. The human genome contains genes encoding separate fractions of tRNA species that function either in the cytoplasm or mitochondria (mt-tRNAs) [83–85]. The complex secondary structure of tRNAs consists of 4 major regions: the amino-acid acceptor stem arm, D arm, T arm, and anticodon [86,87]. The anticodon is a triplicate of nucleotide bases which dictate one of the 20 amino acids to be deposited on the 3’ end of the acceptor stem based on the degenerate coding system. Iso-acceptors, therefore, refer to tRNAs of different sequences, which “accept” the same amino acid during the process of mRNA translation.

The unique structural complexity of this small ncRNA population also translates to a wide array of chemical base modifications that each tRNA may harbor, ranging from as many as 14 to 30 modifications [88,89]. Canonically, tRNAs are critical for protein synthesis and draw the bridging gap from transcriptome to proteome translation, where the degenerate coding system is thought to have been fully explored. However, recent studies have also revealed the cleavage of tRNA species by angiogenin nucleases into smaller fragments – tRFs. tRFs expose sequence motifs that would otherwise be hidden in the uncleaved and folded native structure, and contribute to oncogenesis [90–92].

3.1 miRNA in stem cells

miRNA profiling during human development reveals specific miRNA signatures at various stages, suggesting roles in early lineage differentiation and developmental processes. These small regulatory RNAs play various roles in cancer. miRNAs influence stem cell behavior through several mechanisms (Figure 1A). They regulate the expression of key transcription factors and signaling pathways that govern stem cell maintenance, differentiation, and self-renewal [94,95]. Additionally, miRNAs can modulate the cell cycle, promoting or inhibiting cell division as needed to maintain stem cell populations. They also participate in fine-tuning cellular responses to environmental cues, such as growth factors and cytokines, which further influences stem cell fate decisions. Furthermore, miRNAs are involved in epigenetic regulation by interacting with chromatin-modifying enzymes, thereby affecting the transcriptional landscape of stem cells [96]. Collectively, these mechanisms highlight the pivotal role of miRNAs, explained in further detail below.

Figure 1. Roles of ncRNAs in Stem Cells (A to D) and CSCs (E to H). (A) miRNAs in stem cells. miRNAs play multiple roles in balancing self-renewal and differentiation. They achieve this balance through translational repression and mRNA degradation, controlling the levels of proteins vital for stem cell maintenance and lineage specification. Additionally, miRNAs modulate the cell cycle, ensuring proper progression through different phases essential for self-renewal and differentiation. (B) lncRNAs in stem cells. lncRNAs serve as scaffolds for protein complexes, facilitating the assembly of multi-protein complexes that are essential for various cellular functions. They also play a significant role in epigenetic regulation by interacting with chromatin modifiers to influence gene expression. Additionally, lncRNAs are involved in transcriptional regulation, where they modulate the activity of transcription factors and RNA polymerase. Furthermore, lncRNAs modulate signaling pathways, acting as key regulators of cellular signaling networks. (C) circRNAs in stem cells. circRNAs act as miRNA sponges, sequestering microRNAs and preventing them from binding to their target mRNAs, thereby regulating gene expression. They interact with RNA-binding proteins, influencing the stability, localization, and translation of mRNAs. Additionally, circRNAs are involved in activator/repressor regulation, modulating transcriptional activities. They also serve as targets for methylation, affecting gene expression and cellular functions. (D) tRNAs in stem cells. tRNAs play a fundamental role in protein translation, facilitating the translation of mRNA into proteins. They promote mRNA stability by protecting mRNAs from degradation, thereby ensuring efficient protein synthesis. tRNAs also serve as multi-methylation hotspots, influencing gene expression and cellular functions through epigenetic modifications. Additionally, tRNAs are involved in mitochondrial homeostatic regulation, maintaining mitochondrial function and energy production. (E) miRNAs in cancer cell plasticity. miRNAs facilitate CSC initiation and maintenance, which ultimately drive tumor initiation, growth, and recurrence. They also regulate EMT, enabling cancer cells to gain migratory and invasive properties necessary for metastasis. Moreover, miRNAs promote cancer cell crosstalk and metastasis by facilitating communication between cancer cells and their microenvironment, aiding in metastatic spread and colonization of new tissues. (F) lncRNAs in cancer cell plasticity. lncRNAs regulate alternative splicing, influencing the generation of diverse protein isoforms that contribute to cancer progression. They modulate chromatin architecture, affecting the accessibility of DNA to the transcriptional machinery and thus altering gene expression profiles. lncRNAs also participate in ceRNA networks, where they act as sponges for microRNAs, thereby regulating the availability of microRNAs to their target mRNAs. lncRNAs play a crucial role in reprogramming and dedifferentiation, processes that enable cancer cells to acquire stem cell-like properties and contribute to tumor heterogeneity and adaptability. (G) circRNAs in cancer cell plasticity. circRNAs contribute to apoptotic repression, enabling cancer cells to evade programmed cell death. They enhance the self-renewal capacity of CSCs, promoting tumor growth and recurrence. Additionally, circRNAs are involved in activating cellular signaling pathways that drive cancer progression and metastasis. (H) tRNAs in cancer cell plasticity. tRNAs contribute to the generation of tRFs, which can regulate gene expression and cellular processes. They play a role in cellular metabolic reprogramming, allowing cancer cells to adapt their metabolism to support rapid growth and survival. tRNAs also participate in hormone-dependent regulation, influencing hormone levels and signaling pathways that are crucial for cancer progression.

3.2 lncRNAs in stem cells

Recent literature suggests that close to a thousand lncRNAs have been identified as potential regulators of stem cell renewal [109]. lncRNAs influence stem cell behavior through several key mechanisms (Figure 1B). They act as molecular scaffolds, bringing together protein complexes and directing them to specific genomic locations, thereby modulating gene expression crucial for stem cell maintenance and differentiation. Additionally, lncRNAs can function as decoys, sequestering transcription factors, miRNAs, or other regulatory molecules away from their targets, thus impacting the transcriptional and post-transcriptional landscape of stem cells. They also play roles in chromatin remodeling by interacting with chromatin-modifying enzymes and altering the epigenetic state of stem cell-specific genes. Moreover, lncRNAs can serve as guides, directing chromatin-modifying complexes to specific genomic loci to activate or repress transcription. They also play a role in regulating post-translational modifications by acting as scaffolds. Several of those roles are highlighted below.

3.3 circRNAs in stem cells

Recent literature suggests that numerous circRNAs have been identified that regulate stem cell behavior [119]. circRNAs influence stem cell fate and function through several key mechanisms (Figure 1C). They can act as molecular sponges, sequestering miRNAs and preventing them from repressing their target mRNAs, which can be important for maintaining the balance of gene expression needed for stem cell maintenance and differentiation. Additionally, circRNAs are involved in regulating transcription by interacting with transcription factors and RNA-binding proteins, modulating the transcriptional landscape of stem cells. They also play roles in the splicing process, influencing the production of different isoforms of proteins essential for stem cell functions. Furthermore, circRNAs are implicated in the regulation of apoptosis and cell cycle progression, which are critical for stem cell proliferation and survival. A few examples are provided below.

3.4 tRNAs in stem cells

The structural complexity, diverse sites of modifications, and post-transcriptional modifications of tRNAs present a multitude of factors that contribute to their uniqueness and facilitate biological interplay beyond their traditional role in protein synthesis [128,129]. tRNAs influence stem cell behavior through several key mechanisms (Figure 1D). They play a crucial role in maintaining the cellular protein synthesis machinery, ensuring the production of proteins necessary for stem cell renewal and differentiation. Additionally, tRNAs are involved in regulating gene expression through their interactions with ribosomes and other RNA molecules, impacting the translation efficiency and stability of specific mRNAs. Post-transcriptional modifications of tRNAs, such as methylation and pseudouridylation, can affect their stability and function, thereby influencing stem cell fate decisions. Furthermore, tRNAs can act as signaling molecules, participating in stress response pathways and modulating stem cell survival under adverse conditions. Several examples are detailed below.

4.1 miRNAs

Recent evidence suggests that miRNAs play a regulatory role in cancer cell plasticity [141], impacting processes such as self-renewal, differentiation, and resistance to therapy [142,143] (Figure 1E). They achieve this by modulating gene expression at the post-transcriptional level, which affects the behavior and characteristics of cancer cells. miRNAs can control EMT, a critical process for cancer metastasis, by targeting specific transcription factors and signaling pathways involved in this transition. Additionally, miRNAs influence the TME by altering the expression of genes involved in cell-cell communication and extracellular matrix remodeling. They can be packaged into exosomes and transported to other cells, thereby affecting gene expression and signaling pathways in recipient cells [144]. This crosstalk between cancer cells and their microenvironment facilitated by miRNAs contributes to the dynamic nature of tumor growth and metastasis. Through these mechanisms, miRNAs that are integral to the regulation of cancer cell plasticity are summarized in Table 1, with selected examples elaborated further below.

Table 1. Function of miRNAs in CSCs and their interaction partners

miRNAs function through complex mechanisms that are highly dependent on the specific cell type and molecular context. In glioma stem cells, miR-34a acts as a tumor suppressor by targeting and inhibiting the expression of oncogenes such as c-Met, Notch-1, and Notch-2 [145]. These are key pathways for maintaining the self-renewal and survival of glioma cells. By downregulating these oncogenes, miR-34a induces cell cycle arrest, prevents cell migration, and promotes differentiation, thereby reducing the malignant potential of glioma stem cells. In prostate cancer, miR-34a functions through a different mechanism by directly targeting CD44, a surface adhesion molecule highly expressed in prostate CSCs, which are responsible for tumor initiation, metastasis, and resistance to therapies [146]. miR-34a binds to the 3'-UTR of CD44 mRNA, leading to its degradation and reduced protein expression. The knockdown of CD44 in prostate CSCs phenocopies the effects of miR-34a overexpression, inhibiting clonogenic expansion and metastasis. In vivo studies have shown that systemic delivery of miR-34a significantly reduces metastasis and extends the survival of mice with prostate cancer xenografts. This context-dependent activity of miR-34a underscores the complexity of its regulatory role across various cancers. Its ability to target distinct oncogenes or tumor suppressor genes in different cancer types reflects its potential as a therapeutic tool. This adaptability suggests that its application could be tailored to address the specific molecular pathways that are dysregulated in each cancer type.

miR-31 plays a role in maintaining mammary stem cells and promoting breast cancer development and progression. miR-31 is significantly enriched in breast cancer stem cells (BCSCs) (CD24+CD90+), showing a fivefold increase compared to CD24–CD90– cells [157]. This miRNA favors the expansion of BCSCs and their tumor-initiating capability. Loss of miR-31 reduces metastasis, evidenced by lower levels of EMT-associated factors and metastasis inhibitors, a shift to a mesenchymal-like phenotype, reduced lung metastasis, and higher metastasis-free survival in PyVT/KO mice. Moreover, the expression of miR-31 is regulated by the NF-κB pathway. miR-31 directly targets Wnt antagonists, including AXIN1, GSK3β, and DKK1, thereby activating the Wnt-β-catenin pathway, which ultimately contributes to tumor progression.

The miR-200 family and miR-205 play a crucial role in regulating EMT by targeting the transcriptional repressors ZEB1 and SIP1 [169,170]. These microRNAs are markedly downregulated in cells undergoing EMT, either due to TGF-β exposure or ectopic expression of the protein tyrosine phosphatase Pez. Enforced expression of miR-200 alone can prevent TGF-β-induced EMT, indicating its critical role in maintaining the epithelial phenotype. Conversely, inhibition of these microRNAs induces EMT, characterized by loss of E-cadherin and gain of mesenchymal markers, along with enhanced cell motility. Moreover, the loss of miR-200 and miR-205 is observed in invasive breast cancer cell lines and regions of metaplastic breast cancer specimens, suggesting that mechanisms involved in the downregulation of these microRNAs indeed contribute to tumor progression and metastasis [171].

4.2 lncRNAs

lncRNAs play pivotal roles in regulating cancer cell plasticity through several key mechanisms (Figure 1F) [172]. One of their most well-documented and well-established functions is their role in regulating alternative splicing, leading to diverse protein isoforms that enhance cancer cell adaptability [173–175]. lncRNAs also act as molecular scaffolds, organizing chromatin-modifying complexes and transcription factors at specific genomic loci, thereby altering chromatin architecture to activate or repress gene expression [176–178]. This impacts key cellular processes such as proliferation, differentiation, and the maintenance of CSC properties. Additionally, lncRNAs serve as competing endogenous RNAs (ceRNAs) by sequestering miRNAs, preventing them from binding to their target mRNAs. They further facilitate crosstalk, between cancer cells and the TME, influencing stromal and immune cell behavior to create a supportive niche for tumor growth and metastasis. lncRNAs also promote reprogramming and dedifferentiation, which enhance the stem-like state and resilience of cancer cells. These aspects are detailed in Table 2, with additional examples explored further below.

Table 2. Function of lncRNAs in CSCs and their interaction partners.

In prostate stem-like cells, H19 has been shown to enhance stemness through its regulation of key pluripotency transcription factors, namely OCT4 and SOX2 [184]. Cells expressing high levels of H19 display stem-like properties, including increased expression of stem cell surface markers, resistance to cell death, and enhanced sphere-forming capacity. Suppressing H19 with siRNA leads to a decrease in OCT4 and SOX2 expression, which in turn reduces colony-forming potential and other stem cell-like characteristics. Conversely, overexpression of H19 enhances these traits, promoting the maintenance of prostate CSCs. This suggests that in prostate cells, H19 directly influences transcriptional regulators which govern stemness and survival. In contrast, in BCSCs, H19 plays a different regulatory role through an H19/let-7/LIN28 pathway, highlighting the complexity of lncRNA functions. In this system, H19 acts as a ceRNA to sponge the tumor-suppressive miRNA let-7, which leads to the upregulation of LIN28, a pluripotency factor that supports BCSC maintenance. LIN28 further inhibits the maturation of let-7, creating a feedback loop that reinforces high levels of H19 and LIN28, thereby sustaining BCSC properties such as self-renewal and tumorigenicity. This reciprocal negative feedback loop is essential for maintaining BCSCs and promoting their therapy resistance [185]. These examples underscore the context-dependent role of lncRNAs.

The lncRNA MALAT1 plays a functional role in regulating cell cycle progression by modulating the expression of the oncogenic transcription factor B-MYB. MALAT1 is differentially expressed throughout the cell cycle and is essential for both the G1/S and mitotic phases. Depletion of MALAT1 activates p53 and its target genes, leading to cell cycle arrest and reduced cellular proliferation. This cell cycle arrest is particularly noteworthy as it implicates p53 as a major downstream mediator of MALAT1 activity. Furthermore, MALAT1 promotes the proliferation and invasiveness of CSCs in pancreatic and liver cancers by modulating the expression and pre-mRNA processing of key transcription factors involved in mitotic progression [186,199]. MALAT1-depleted cells also exhibit defects in chromosome segregation and spindle assembly, highlighting MALAT1’s role in maintaining genomic stability during mitosis [200].

lncRNA HOTAIR interacts with the Polycomb Repressive Complex 2 (PRC2) to modify histones and silence gene expression, affecting EMT and CSC maintenance [187]. In BCSCs, HOTAIR indirectly suppresses miR-7, which normally inhibits the oncogene SETDB1. This suppression facilitates EMT and the maintenance of the CSC pool by upregulating the STAT3 pathway, leading to increased expression of c-myc, Twist, and miR-9 [188]. Moreover, HOTAIR modulates the expression of HoxD10, which contributes to miR-7 downregulation and further promotes tumor invasiveness.

lncRNA SOX2 Overlapping Transcript (SOX2-OT) regulates the expression of the SOX2 gene, a critical factor for maintaining pluripotency and self-renewal in stem cells. This lncRNA enhances the stem-like properties of cancer cells by upregulating SOX2 expression. SOX2-OT is involved in various cancers, where it promotes tumor growth and CSC maintenance by regulating SOX2 levels. This regulation contributes to the aggressiveness and recurrence of these malignancies [190,191,201].

4.3 circRNAs

circRNAs contribute to the plasticity of cancer cells under stress and therapeutic pressures through several mechanisms (Figure 1G) [202]. In cancer cells, circRNAs act as molecular sponges for miRNAs to prevent the repression of oncogenic targets, thus promoting cancer progression and metastasis. Additionally, circRNAs interact with RNA-binding proteins and transcription factors to modulate gene expression and stabilize mRNAs related to cancer, leading to changes in cell cycle progression, apoptosis resistance, and enhanced metastatic potential. This modulation of gene expression primarily supports cell survival and proliferation in cancer. Furthermore, circRNAs in cancer cells facilitate dynamic adaptability by regulating apoptosis and cell cycle pathways, enabling cancer cells to evade death and sustain growth. These circRNAs are detailed in Table 3 and further explained below.

Table 3. Function of circRNAs in CSCs and their interaction partners.

The circRNA circAGFG1 was found to be upregulated in colorectal cancer cell lines and its knockdown leads to a marked reduction in cell proliferation, migration, invasion, and stemness, while enhancing apoptosis [205]. This indicates the crucial role of circAGFG1 in maintaining the CSC properties, including self-renewal and metastatic potential. The mechanism involves circAGFG1 sponging miR-4262 and miR-185-5p, which ordinarily suppress the expression of the transcription factor YY1. By sequestering these miRNAs, circAGFG1 facilitates the upregulation of YY1, which in turn promotes the transcription of CTNNB1. The activation of the Wnt/β-catenin signaling pathway through this circAGFG1/miR-4262/miR-185-5p/YY1/CTNNB1 axis enhances CSC properties and contributes to colorectal cancer progression and metastasis.

The circRNA hsa_circ_002178 is significantly upregulated in breast cancer tissues and cell lines and its high expression correlates with poor clinical outcomes, including low survival rates, larger tumor size, lymph node metastasis, and advanced TNM stage. Mechanistically, hsa_circ_002178 promotes the growth, invasion, and migration of breast cancer cells by modulating the miR-1258/KDM7A axis. Overexpression of hsa_circ_002178 leads to the formation of microspheres, and increased levels of stemness markers (ALDH1, CD44, NANOG, and OCT4), and elevated ALDH1 activity, which are indicative of enhanced CSC properties. This circRNA acts as a sponge for miR-1258, thereby reducing the miRNA's inhibitory effect on KDM7A. KDM7A, in turn, facilitates the stem-like characteristics of breast cancer cells, contributing to tumor growth and metastasis [206].

The circRNA circCTIC1 was found to be highly expressed in colon tumors and colon tumor-initiating cells (TICs), and its knockdown impaired the self-renewal capacity of these cells, whereas overexpression enhanced it. Mechanistically, circCTIC1 interacts with the nuclear remodeling factor (NURF) complex, recruiting it to the c-Myc promoter, thereby facilitating the transcriptional activation of c-Myc, a core transcription factor for stemness. This interaction underscores the role of circCTIC1 as a protein modulator, driving the self-renewal and tumorigenic potential of colon TICs through the NURF-mediated c-Myc pathway [207].

4.4 tRNAs

tRNAs in cancer cells can undergo extensive modifications, enhancing their stability and functionality to meet the increased demands for protein synthesis of rapidly proliferating cancer cells (Figure 1H). These modifications can affect the efficiency and accuracy of translation, promoting the production of proteins that support cancer progression and metastasis [212]. Additionally, tRFs have recently emerged as novel regulators in cancer, where they can act as signaling molecules, influencing gene expression, and modulating cellular stress responses. tRNAs and tRFs are often co-opted to support oncogenic processes, including the regulation of apoptosis, cell cycle progression, and metabolic reprogramming, enabling cancer cells to evade cell death, sustain growth, and adapt to hostile microenvironments. Thus, tRNAs in cancer cells play a multifaceted role in maintaining the plasticity and resilience of cancer cells, distinguishing their function from the more static roles they perform in normal cellular contexts. tRNAs affecting cancer cell plasticity are detailed in Table 4, with some examples highlighted below.

Table 4. Function of tRNAs in CSCs and their interaction partners.

tDR-000620 has been identified as significantly downregulated in the CSCs of TNBC. tDR-000620 was found to play a pivotal role in the suppression of CSC traits, including the inhibition of EMT, recurrence, and metastasis. In TNBC, where the CSC population is highly enriched, the downregulation of tDR-000620 correlates with enhanced self-renewal and tumor-initiating capacities, driving aggressive tumor behavior. Moreover, low tDR-000620 expression was shown to be an independent adverse prognostic factor for recurrence-free survival, further linking its role to poor clinical outcomes. Functional analyses suggest that tDR-000620 is involved in immune-response pathways such as cytokine-cytokine receptor interactions and AMPK signaling, which are essential in the regulation of cell proliferation, metabolism, and stress responses [213].

tRF/miR-1280 has emerged as a key regulator of CSC-like properties and metastasis in colorectal cancer (CRC). This 17-base pair fragment was shown to suppress Notch signaling pathways that support the function and maintenance of CSCs, which are crucial for tumor progression and metastasis. tRF/miR-1280 is significantly downregulated in CRC tissues compared to adjacent normal tissues, and its ectopic expression reduces cell proliferation, colony formation, and metastasis, indicating a tumor-suppressive function. Mechanistically, tRF/miR-1280 directly targets the JAG2 ligand, inhibiting the activation of Notch1 and Notch2 receptors, which are essential for sustaining CSC-like traits in CRC. Through the inactivation of Notch signaling, tRF/miR-1280 leads to the downregulation of critical transcription factors like Gata1 and Gata3, and it increases the expression of miR-200b, a key regulator of EMT. This cascade ultimately results in the repression of ZEB1 and SUZ12, key drivers of CSC maintenance and EMT, thus preventing tumor growth and metastasis [215].

Glycine tRNA-derived fragment (Gly-tRF) enhances liver CSC-like properties and promotes the migration of hepatocellular carcinoma (HCC) cells by targeting NDFIP2, a key regulator of ubiquitin ligase function. Gly-tRF is upregulated in HCC tissues and cell lines, where it drives EMT and increases the stemness and migratory capacity of cancer cells. Mechanistically, Gly-tRF downregulates NDFIP2 by binding to its mRNA 3′-UTR, thereby activating the AKT signaling pathway, a pathway known to contribute to tumor progression and metastasis [216].

5.1 Biomarkers and diagnostics

5.2 Targeting methods and delivery

5.2.1 miRNAs

Oncogenic miRNA expression can be effectively inhibited using antisense oligonucleotides (ASOs), which bind to miRNA targets via base-pair complementarity (Figure 2A). The function of ASOs is enhanced by incorporating various chemical configurations such as locked nucleic acids, anti-miRNA oligonucleotides, and antagomirs, which improve their stability and efficacy [243,244]. For instance, an antagomir specifically designed to target and knockdown the oncogene miR-21 has been employed in breast cancer MCF-7 cells. This intervention resulted in a notable reduction of MCF-7 tumor growth in both in vitro and in vivo tumor xenograft models, achieved by inhibiting cell proliferation and inducing apoptosis [245].

Figure 2. Therapeutic Potentials of ncRNAs. (A) Strategies for targeting oncogenes and tumor suppressor genes using miRNAs. For oncogenes, synthetic miRNA mimics and lentiviral miRNA transfection can inhibit oncogenic pathways. In contrast, for tumor suppressor genes, miRNA-targeted RNA sponges and ASOs can enhance the expression of these genes by sequestering miRNAs that would otherwise repress them. (B) CRISPR-based approaches for regulating oncogenes and tumor suppressor genes through lncRNAs. Oncogenes can be targeted using dCas9-KRAB CRISPR interference (CRISPRi) to inhibit gene expression or Cas9-dsDNA mediated cleavage for gene disruption. Tumor suppressor genes can be activated using dCas9-VP64 CRISPR activation (CRISPRa) to enhance their expression via lncRNAs. (C) Methods for targeting circRNAs in cancer therapy. Synthetic circRNA sponges can sequester miRNAs, while Cas13 ssRNA-mediated back-splice junction targeted cleavage and siRNA-harboring nanoparticles can lead to circRNA degradation through the RISC complex, reducing their oncogenic functions. (D) Approaches for modulating tRNA functions in cancer cells. Inhibition of methyltransferase proteins using small molecule inhibitors can prevent the methylation of tRNAs, thereby disrupting protein translation. Additionally, ASOs can bind to pre-mature tRNAs, preventing their maturation and function in protein synthesis.

Additionally, the suppression of oncogenic miRNAs can be achieved using “miRNA sponges” (Figure 2A). These sponges work by incorporating complementary binding sites for miRNA-targeted RNA into RNA transcripts expressed from strong promoters. This mechanism effectively sequesters miRNAs, preventing them from binding to their endogenous targets, thereby inhibiting miRNA activity in cultured cancer cells and mouse models [243]. Inhibiting miR-22 with a specific “sponge” in LM2 cells, a highly metastatic breast cancer cell line, led to a significant decrease in breast cancer metastasis to the lungs [246]. In contrast, miRNA mimics or lentiviral vectors can be employed to elevate the expression levels of tumor suppressor miRNAs, which are typically found to be underexpressed in cancer cells [247]. RNA interference (RNAi) includes siRNAs and short hairpin RNAs (shRNAs). While siRNAs offer transient gene silencing due to their instability, shRNAs provide durable and long-lasting effects in vivo [248,249]. Virus transfection, the primary method for delivering shRNA plasmids to target sites, can also be employed to introduce exogenously synthesized miRNA plasmids into cancer cells, thereby upregulating the corresponding miRNA.

5.3 Potential toxicity

Ethics Statement

Not applicable.

Consent for Publication

Not applicable.

Funding

This work was supported by the Princess Margaret Cancer Foundation (886012001223 to H.H.H.), CIHR operating grants (142246, 152863, 152864 and 159567 to H.H.H.), Terry Fox New Frontiers Program Project Grant (PPG19-1090 and PPG23-1124 to H.H.H.). H.H.H. holds Tier 1 Canada Research Chair in RNA Medicine. P.H. was supported by a MOHCCN Health Informatics Award and a CIHR Doctoral Award.

Competing Interests

The authors have declared that no competing interests exist.