{"paper_id":"41f09ea7-4e4d-43d5-b327-91f84275093d","body_text":"As an evolutionarily conserved intercellular communication system, the Notch signaling pathway serves crucial roles in cell differentiation, proliferation, apoptosis, and stem cell maintenance. \n 1 \n  This pathway is regarded as a pillar of juxtacrine signaling that arranges complex intercellular communication, controlling different developmental and homeostatic processes through a firmly orchestrated cascade of enzymatic cleavage reactions. \n 1 \n  This pathway results in the nuclear transposition of the Notch intracellular domain (NICD) and the successive induction of downstream target genes. Four Notch receptors (Notch 1, Notch 2, Notch 3, and Notch 4) \n 2 \n  and five ligands for these receptors (Dll‐1, Dll‐3, Dll‐4, Jagged 1, and Jagged 2) \n 3 \n  have been identified in humans. The interaction between Notch receptors and their ligands results in the activation of the Notch pathway. In fact, this interaction triggers proteolytic cleavage by γ‐secretase, releasing the NICD, which enters the nuclear compartment. \n 1 \n  Subsequently, the NICD cooperates with CSL (CBF1/RBP‐Jκ, Su(H), Lag‐1) transcription factors, recruiting coactivators like Mastermind‐like (MAML) to activate expression of a number of targets, including Hes and Hey family members. \n 1 \n  Figure  1  illustrates the key steps of how Notch receptors are activated and how they trigger downstream signaling events.\nThe key steps of the activation of Notch receptors and how they trigger downstream signaling events.\nThese genes have a crucial impact on a variety of molecular processes, such as cell cycle transition, cellular differentiation, intercellular adhesion, and commitment to specialized cell fates. \n 4\nAberrant function of Notch signaling is associated with various disorders, including cancer, \n 5 \n  cardiovascular disorders, \n 6 \n  neurodegenerative conditions \n 7 \n , and reproductive disorders. \n 4\nIn recent years, circular RNAs (circRNAs) have been acknowledged as key regulators of gene expression. \n 8 \n  These single‐stranded, covalently closed RNA molecules are extremely stable and display a tissue‐specific expression pattern. \n 9 \n  Growing evidence proposes that circRNAs have a significant role in modulating the Notch pathway mainly by acting as microRNA (miRNA) sponges. \n 10 \n  Moreover, other mechanisms such as their interaction with RNA‐binding proteins (RBPs), \n 11 \n  or even production of functional peptides might contribute to the regulation of certain signaling pathways. \n 12\nThis article explores the mechanisms through which circRNAs regulate the Notch pathway and their implications in disease and therapy.\n\nCirc‐NOTCH 1 is an upregulated circRNA in gastric cancer. Notably, circ‐NOTCH 1 has multiple binding sites for miR‐637. Additionally, this miRNA targets apelin, another gene that is highly expressed in gastric cancer cells and tissues. Circ‐NOTCH 1 increases cell proliferation and invasiveness, and decreases cell apoptosis through inhibiting the transcriptional activity of miR‐637, thus increasing apelin levels. \n 13 \n  Similarly, circ‐NOTCH 1 antagonizes miR‐449c‐5p availability to facilitate MYC‐induced NOTCH 1 overexpression that eventually leads to induction of metastasis and stem cell properties in gastric cancer. \n 14 \n  Besides, c‐Myc‐induced circ‐NOTCH 1 enhances aggressiveness of nasopharyngeal carcinoma cells through modulating the miR‐34c‐5p/c‐Myc axis. \n 15\ncircNFIX is another circRNA that is overexpressed in glioma tissues compared with corresponding normal tissue samples consistent with upregulation of the Notch signaling pathway. Notably, circNFIX acts as a sponge for miR‐34a‐5p, an miRNA that targets NOTCH 1. Both silencing of circNFIX and overexpression of miR‐34a‐5p inhibit cell propagation and migration. Besides, an miR‐34a‐5p inhibitor could neutralize the inhibitory effect of siRNA designed against circNFIX (si‐circNFIX) on glioma cells. As in vivo assays show, si‐circNFIX inhibits glioma growth through regulation of miR‐34a‐5p and NOTCH 1. \n 16\nExpression of NOTCH 1 and hsa_circ_0005986 as a NOTCH 1 regulatory circRNA has been affected by an extremely low‐frequency magnetic field (ELF‐MFs). Notably, MFs could decrease the viability of gastric tumor cells, while increasing the viability human normal fibroblast. Besides, all magnetic flux densities (MFDs) have downregulated NOTCH 1 in gastric tumor cells and upregulated its expression in normal fibroblasts, dependent on the MFD of MFs. In fact, the tumor and normal cells have exhibited distinct molecular behavior in different MFDs regarding NOTCH 1 and hsa_circ_0005986 levels. Reduction of tumor cells survival after exposure to ELF‐MFs might be due to downregulation of NOTCH 1 and hsa_circ_0005986. \n 17\nA high throughput circRNA analysis has resulted in the identification of circRNA‐000911 as a downregulated circRNA in breast cancer cells. \n 18 \n  Functional studies have shown that overexpression of circRNA‐000911 inhibits proliferation, migration, and invasion, and promotes apoptosis. \n 18 \n  RNA precipitation assay has suggested miR‐449a as the circRNA‐000911‐associated miRNA. Mechanistically, miR‐449a antagonizes circRNA‐000911 to affect progression of breast cancer. \n 18 \n  Most notably, Notch 1 is a target of miR‐449a, through which circRNA‐000911 influences breast cancer progression. Besides, NF‐κB signaling is functionally influenced by the circRNA‐000911/miR‐449a axis. \n 18 \n  Taken together, circRNA‐000911 has an anti‐oncogenic effect in breast cancer and might be used as a therapeutic target in this cancer. \n 18\nOriginated from exons 1 and 2 of the  NSD2  gene, circ‐NSD2 is overexpressed in colorectal cancer tissues, particularly in advanced stages or metastatic tumors. \n 19 \n  This circRNA increases the migration, invasiveness, and metastasis of cancer cells through targeting miR‐199b‐5p and subsequent regulation of DDR1 and Jagged 1. \n 19 \n  The latter is regarded as a ligand for NOTCH 1 and is implicated in epithelial mesenchymal transition in colorectal cancer. \n 20 \n  In fact, DDR1 and Jagged 1 have a synergic effect on regulation of cell–matrix interactions, migration, and metastasis of colorectal cancer. \n 19 \n  Table  1  shows circRNAs that affect NOTCH 1 expression in different cancers.\nRole of circRNAs in the regulation of NOTCH 1 expression in cancers.\ncircKIF4A (hsa_circ_0007255) is an example of circRNAs contributing to the development and progression of bladder cancer through modulating NOTCH 2 expression. Its expression has been significantly enhanced in clinical samples and cell lines originated from this type of cancer. Notably, its knockdown has led to inhibition of the proliferation and colony formation capacity of bladder cancer cells. In addition, migration and metastatic aptitude of cancer cells have been dramatically reduced after circKIF4A silencing. This circRNA sponges miR‐375/1231 to stimulate bladder cancer progression through enhancing expression of NOTCH 2. \n 32 \n  Table  2  shows circRNAs that affect NOTCH 2 expression in different cancers.\nRole of circRNAs in the regulation of NOTCH 2 expression in cancers.\nCirc_0000043 (circ_PUM1) is an example of circRNAs contributing to the progression of endometrial cancer through regulation of NOTCH 3 expression. This circRNA has been expressed at expressively higher level in endometrial cancer samples than in normal samples. Its upregulation promotes proliferation, migration, and invasion of endometrial carcinoma cells. Mechanistically, circ_PUM1 binds to miR‐136, thus increasing its target gene NOTCH 3. These effects can be inverted by upregulation of miR‐136, suggesting the functional role of circ_PUM1/miR‐136/NOTCH3 axis in the development of endometrial cancer. \n 34 \n  CircRNA‐mediated modulation of NOTCH 3 is also involved in the pathogenesis of esophageal squamous cell carcinoma (ESCC). Being upregulated in ESCC tissues, hsa_circ_0001741 is positively associated with lymphatic metastasis, high TNM stage, and poor clinical outcome. Functionally, hsa_circ_0001741 stimulates cancer stemness, invasion, and migration through sequestering the tumor suppressor miRNA miR‐491‐5p. Precisely, hsa_circ_0001741 binds to miR‐491‐5p to preclude its binding to the 3′‐UTR of NOTCH 3 transcript and inhibiting NOTCH 3 expression. Furthermore, hsa_circ_0001741 silencing significantly inhibits the in vivo tumorigenic properties of ESCC cells. Taken together, hsa_circ_0001741 is a potential prognostic marker for ESCC. \n 35 \n  Table  3  shows circRNAs that affect NOTCH 3 expression in different cancers.\nRole of circRNAs in the regulation of NOTCH 3 expression in cancers.\nCircRNA‐mediated regulation of Notch signaling is also involved in the pathogenesis of a variety of nonmalignant disorders (Table  4 ), such as preeclampsia (PE), \n 42 \n  neuropathic pain \n 43 \n , endometriosis, \n 41 \n  Alzheimer's disease (AD), \n 49 \n  viral myocarditis, \n 45 \n  and osteoarthritis (OA). \n 37 \n  For instance, being upregulated in PE placentas compared to normal pregnancy placenta, hsa_circ_0111277 level is positively correlated with PE‐related parameters. This circRNA is mainly localized within the cytoplasm of trophoblasts where it sponges hsa‐miR‐494‐3p to weaken the repressive effect of this miRNA on HTRA1/Notch 1 levels. In fact, migration and invasion of trophoblasts are regulated by the hsa_circ_0111277/miR‐494‐3p/HTRA1/Notch 1 axis, potentiating this axis as a new therapeutic target for PE. \n 42 \n  Additionally, function of circ_0005075 in neuropathic pain has been evaluated in the mice model of chronic constriction injury (CCI) mimicking neuropathic pain in human subjects. \n 43 \n  Being appreciated as a key oncogene in several malignancies, \n 52 \n ,  \n 53 \n  it is also regarded as an inflammation‐associated circRNA. Expression of circ_0005075 has been evidently elevated in CCI rat models. Silencing of circ_0005075 has suppressed mechanical and thermal hypersensitivity to pain. Furthermore, circ_0005075 knockdown has repressed the neuroinflammation through targeting COX‐2, IL‐6, and TNF‐α, increasing IL‐10 levels and modulating NOTCH 2. Additional experiments have shown that miR‐151a‐3p is a target of circ_0005075 that is decreased in CCI rats. Taken together, loss of circ_0005075 has reduced neuropathic pain through induction of miR‐151a‐3p and suppression of NOTCH 2. \n 43\nInvolvement of circRNAs in the modulation of the Notch pathway in malignant disorders.\nCircRNA‐mediated regulation of the Notch pathway is also implicated in the pathogenesis of endometriosis. Notably, expression of hsa_circ_0067301 and miR‐141–5p has been found to be decreased in ectopic endometrium compared to control endometrial samples. Knockdown of hsa_circ_0067301 has led to enhancement of proliferation and migration of Ishikawa and End1/E6E7 cells, along with elevation of expression of Notch 1, Hes‐1, N‐cadherin, and vimentin but reduction of E‐cadherin levels. In fact, hsa_circ_0067301/miR‐141–5p/Notch 1 axis has been shown to exert a crucial regulatory role in the process of epithelial–mesenchymal transition in endometriosis. \n 41\nWhile the abovementioned circRNAs have a relatively direct effect on Notch signaling, circ_0004381 affects expression of presenilin 1 (PSEN1), \n 49 \n  a key protein involved in the γ‐secretase complex, which is essential for the cleavage of Notch receptors, a process crucial for Notch signaling. \n 54 \n  Considering the importance of PSEN1 in the pathogenesis of AD through regulation of the Notch pathway, \n 55 \n  it is not surprising that circ_0004381 has a key role in the pathogenesis of AD. In fact, expression of this circRNA has been found to be elevated in Aβ1‐42‐treated hippocampal neurons. Circ_0004381 silencing has alleviated Aβ1‐42‐triggered apoptosis, oxidative damage, and mitochondrial impairment in hippocampal neurons. Moreover, its knockdown has enhanced microglial M2‐type polarization, suppressed the release of inflammatory molecules by microglia, and improved cognitive functions in animal models of AD. From a mechanistical point of view; circ_0004381 regulates PSEN1 expression through sequestering miR‐647, suggesting the importance of the circ_0004381/miR‐647/PSEN1 axis in the pathogenesis of AD through modulation of the Notch pathway. \n 49\nAdditionally, hsa_circ_0063331 (circDDX17) is involved in the pathoetiology of coxsackievirus B3‐induced myocarditis through regulation of NOTCH 2. Expression of this circRNA has been considerably reduced after infection with this virus. Moreover, circDDX17 enhances replication of viral particles through decreasing the expression of miR‐1248, an miRNA that interacts with NOTCH 2. In this context, NOTCH 2 upregulates expression of methyltransferase‐like protein 3, a protein that regulates viral replication. \n 45\nFinally, circ_0104873 has been identified as an upregulated circRNA during bone marrow mesenchymal stem cells (BMSCs) differentiation toward osteogenic lineage. Mechanistically, circ_0104873 sponges miR‐875‐5p to increase expression of NOTCH 3, thus stimulating the Notch signaling pathway. Notably, this circRNA contributes to the development of OA through modulating the miR‐875‐5p/NOTCH 3/Notch axis. \n 37\n\nPersonalized medicine has been emerged as a novel modality to precise and targeted therapy of cancer. \n 56 \n  This field involves modulation of certain signaling pathways as well as the noncoding region of the genome. \n 57 \n  A practical strategy to inhibit the development and spread of tumors is to interfere with the Notch signaling pathways. Novel approaches to specifically hinder pro‐tumorigenic functions of this pathway can be developed through modulation of the function of circRNAs. Thus, circRNAs that regulate the Notch pathway have therapeutic potential in cancer. In the vast majority of cases, overexpression of oncogenic circRNAs enhances Notch signaling, promoting tumor growth. Conversely, tumor‐suppressive circRNAs inhibit Notch‐driven malignancies. However, there are some exceptions to this rule. For instance, the oncogenic circRNA, circ_0008532 enhances bladder cancer cells migration, invasion, and angiogenesis through inhibiting the Notch signaling pathway. \n 28 \n  It is worth mentioning that the Notch pathway can exert both oncogenic and tumor suppressor functions depending on cell context as well as the genetic landscape inside a particular cell type. \n 58 \n  In fact, the Notch pathway's functional duality is orchestrated by circRNAs in a tissue‐ and disease‐specific manner. CircRNA stoichiometry and competing endogenous RNA (ceRNA) networks may determine Notch's role, with high circRNA levels favoring oncogenic outcomes in proliferative tissues.\nA recurring theme across both cancers and nonmalignant diseases is the sequestration of tumor‐suppressive miRNAs by circRNAs, leading to derepression of Notch signaling. Key examples include miR‐34 and miR‐449 families. CircNFIX and circ‐ASH2L sponge miR‐34a‐5p, elevating NOTCH 1 expression and driving proliferation of glioma \n 16 \n  and pancreatic cancer cells, \n 22 \n  respectively. Circ‐NOTCH 1 (gastric cancer) \n 14 \n  and circ‐000911 (breast cancer) \n 18 \n  antagonize miR‐449c‐5p and miR‐449a, respectively, amplifying NOTCH 1 activity and metastasis. These conserved miRNA–circRNA interactions suggest that therapeutic targeting of specific miRNA nodes (e.g., miR‐34 or miR‐449) could simultaneously disrupt Notch signaling in diverse pathologies.\nWhile the role of NOTCH 1‐interacting circRNAs has been more assessed in the pathogenesis of cancers, circRNAs that interact with other NOTCH receptors or ligands have been less studied. Figure  2  shows an overview of circRNAs that regulate different Notch receptors.\nOverview of circRNAs that regulate different Notch receptors through miRNA sponging.\nDespite tissue‐specific contexts, circRNA‐mediated Notch activation converges on common downstream effectors. For instance, DLL/JAG ligands are examples of these downstream effectors. Circ‐NSD2 upregulates JAG1 in colorectal cancer, \n 19 \n  while circ_0006476 modulates DLL4 in atherosclerosis, \n 39 \n  illustrating how circRNAs regulate Notch ligands to amplify pathway activity across diseases. Targeting these downstream effectors (e.g., with γ‐secretase inhibitors) may circumvent tissue‐specific circRNA heterogeneity.\nCircRNAs are emerging as important regulators of the Notch pathway through miRNA sponging, interaction with some proteins, and perhaps in rare cases, peptide encoding. However, the latter function has not been well‐established in this context. Their dysregulation contributes to various diseases, particularly cancers; thus, they are attractive targets for therapy.\nCurrent research on NOTCH‐interacting circRNAs has predominantly focused on their oncogenic or tumor‐suppressive roles in cancer. However, their implications in nonmalignant conditions remain underexplored. To date, only a handful of disorders have been investigated in this context, including PE, \n 42 \n  neuropathic pain \n 43 \n , endometriosis, \n 41 \n  AD, \n 49 \n  viral myocarditis, \n 45 \n  and OA. \n 37 \n  The narrow scope of studied diseases highlights a significant knowledge gap, particularly in cardiovascular, metabolic, and autoimmune disorders. Besides, most studies have correlated circRNA expression changes with disease states in animal models or cell lines but lack validation in human samples. Whether targeting NOTCH‐interacting circRNAs could ameliorate these conditions remains unexplored. In fact, expanding research into nonmalignant diseases could uncover novel circRNA‐dependent processes and therapeutic prospects associated with the NOTCH pathway.\nIt is worth mentioning that circRNAs exploit Notch signaling to drive hallmark cancer phenotypes, which overlap with nonmalignant disorders. EMT and metastasis are important examples in this regard. Circ‐NSD2 (colorectal cancer) \n 19 \n  and hsa_circ_0067301 (endometriosis) \n 41 \n  both regulate Notch to modulate expressions of E‐cadherin and N‐cadherin/vimentin.\nStemness is another feature shared between these two conditions that might be subject of circRNA‐mediated regulation of the Notch pathway. Besides, angiogenesis as another important shared feature between malignant and nonmalignant conditions has been shown to be affected by circ_0084582 and circHipk3 in the contexts of osteosarcoma \n 29 \n  and myocardial infarction, \n 46 \n  through modulation of VEGF and DLL4, respectively. These shared hallmarks suggest that circRNA–Notch interactions are not disease‐specific but rather represent universal regulatory modules adaptable to different pathological contexts.\nThe summarized data in this article show that synthetic circRNAs or antisense oligonucleotides can be designed to modulate Notch signaling. For instance, silencing circNFIX in glioma models has dramatically reduced tumor growth via miR‐34a‐5p/NOTCH 1 inhibition. \n 16 \n  Besides, overexpression of circ_000911 has suppressed breast cancer xenografts by restoring the miR‐449a/Notch 1 axis. \n 18\nSmall molecules or antisense oligonucleotides against recurrent miRNA sponges (e.g., miR‐34a‐5p) could disrupt multiple circRNA–Notch axes. Moreover, combination of γ‐secretase inhibitors with circRNA‐targeting therapies may enhance efficacy. However, challenges remain in delivering circRNA modulators selectively to tumor or diseased tissues without off‐target effects. Since naked circRNAs are degraded rapidly, exosome‐encapsulated circRNAs or lipid nanoparticles can be used for their delivery. A possible source for off‐target effects is miRNA pleiotropy. Application of tissue‐specific promoters or CRISPR‐based circRNA editing might be regarded as possible solutions for this obstacle. Future research should focus on translating these findings into clinical applications, offering new avenues for personalized medicine.\nBesides, the discovery of circRNAs linked to tumors caused by dysregulation of the Notch pathway may provide imperative means for early diagnosis and prognosis. Research has shown that a number of these circRNAs, such as hsa_circ_0001741 could serve as prognostic markers in cancer. \n 35 \n  Additionally, hsa_circ_0005986** has exhibited prognostic value in hepatocellular carcinoma; \n 30 \n  however, research is needed to evaluate its abundance in biofluids and introduce ultrasensitive detection methods for its quantification. Similarly, hsa_circ_0001741 levels correlate with advanced stage and poor survival in ESCC, supporting its use as liquid biopsy target. \n 35 \n  The prognostic or diagnostic prospects for other circRNAs should be uncovered in future studies.\nFinally, it is worth mentioning that virtually all covered interactions described in this review were restricted to the miRNA sponging activity of circRNAs, neglecting other functions of circRNAs (e.g., protein scaffolding, alternative splicing regulation, or peptide translation). Future studies should also consider these less‐characterized yet important activities of circRNAs. Moreover, it is necessary to assess whether certain circRNAs can affect Notch ligand trafficking or post‐translational modifications. Another unexplored area in the field of circRNAs effects on the Notch pathway is the interaction between circRNAs and natural therapeutic compounds. Recently, natural compounds and proteasome targeting drugs have been suggested as possible treatment modalities for cancer. \n 59 \n ,  \n 60 \n ,  \n 61 \n  Thus, the effects of these compounds on Notch‐modulating circRNAs should be investigated in future studies.\n\nDevelopment of targeted therapies to interfere with cancer‐promoting function of Notch is a feasible option in the dawn of precision medicine. Interference with circRNAs that regulates this pathway is among treatment alternatives that specifically target indispensable constituents of the Notch signaling system. A more ample understanding of the spatiotemporal evolving patterns of Notch activity within the tumor niche maybe acquired through incorporating advanced sequencing methods such as single‐cell sequencing and functional assays. Considering the NOTCH's druggability (e.g., γ‐secretase inhibitors), \n 62 \n  future efforts should focus on translational studies to unravel the clinical relevance of dual manipulation of the Notch pathway with such drugs and circRNA‐targeting strategies.\nWhile the role of circRNAs in Notch regulation is increasingly recognized, challenges remain. More studies are required to clarify specific circRNA–Notch interactions. Moreover, in vivo models are required to confirm therapeutic potential of manipulation of circRNAs. Finally, developing circRNA‐based drugs needs overcoming delivery and stability issues.\n\nPegah Yazdanpanah searched the literature, and designed tables and figures. Amir Sadeghi contributed to the study design and supervision. Soudeh Ghafouri‐Fard supervised the study, wrote the manuscript, and revised it.\n\nThe authors declare no conflicts of interest.\n\nNot applicable (no animal or clinical study).","source_license":"CC-BY-4.0","license_restricted":false}