Circ_0003945: an emerging biomarker and therapeutic target for human diseases

Due to the rapid development of RNA sequencing techniques, a circular non-coding RNA (ncRNA) known as circular RNAs (circRNAs) has gradually come into focus. As a distinguished member of the circRNA family, circ_0003945 has garnered attention for its aberrant expression and biochemical functions in human diseases. Subsequent studies have revealed that circ_0003945 could regulate tumor cells proliferation, migration, invasion, apoptosis, autophagy, angiogenesis, drug resistance, and radio resistance through the molecular mechanism of competitive endogenous RNA (ceRNA) during tumorigenesis. The expression of circ_0003945 is frequently associated with some clinical parameters and implies a poorer prognosis in the majority of cancers. In non-malignant conditions, circ_0003945 also holds considerable importance in diseases pathogenesis. This review aims to recapitulate molecular mechanism of circ_0003945 and elucidates its potential as a diagnostic and therapeutic target in neoplasms and other diseases.


Introduction
Cancer represents a serious public health issue that affects people all around the world (1).Various etiological factors and socioeconomic elements including population aging exacerbate the cancer burden and contributing to an increase in cancer-related fatalities (2).Despite the availability of cutting-edge medical diagnostics and therapeutics, recurrence and metastasis still pose dramatic barriers to achieving long-term survival in patients (3)(4)(5).
CircRNAs, a new class of non-coding RNA (ncRNA) distinct from traditional linear RNAs, are characterized by their covalently closed, continuous loop structures (6).First identified in plants viroids in 1976 (7), circRNAs were subsequently detected in eukaryotic cells in 1979 (8).The discovery that the hepatitis delta virus (HDV) has a single-stranded circRNA molecule marked it as the first known animal virus with a circRNA genome in 1986 (9).Specifically, circRNA is generated by the RNA polymerase II (pol II)-mediated back-splicing of pro-mRNA (10).Back-splicing forms a stable closed-loop structure devoid of 3' or 5' end.It restrains the exonuclease-mediated degradation due to its covalent bonding of downstream and upstream splice-donor sites (11).The nucleotide (nt) length of circRNAs typically ranges from several hundred to a few thousand, and it generally composed of one to five exons (12).Depending on their splicing sources, circRNAs can be categorized into single-exonic circRNAs, exonic circRNAs (EcircRNAs), intronic circRNAs (CiRNAs), exon-intron circRNAs (EIciRNAs), even tRNA intron cirRNAs (tricRNAs) according to their splicing sources (Figure 1) (13)(14)(15).CircRNAs are widely expressed in eukaryotes, where EcircRNAs are predominantly localized in the cytoplasm, while EcircRNAs and EIciRNAs are primarily nucleus (16,17).In summary, circRNAs demonstrates tissue-specific localization and evolutionary conservation, stably persisting in intricate intracellular and extracellular environments, holding promise as ideal tumor biomarkers (18,19).CircRNAs perform various biological functions (Figure 2): (1) as endogenous RNAs (ceRNA), circRNAs act as molecular sponges, impeding the microRNA (miRNA)-mediated repression of target genes (20,21).(2) CircRNAs serve as molecular sponges for proteins, particularly RNA-binding proteins, regulating the transcription or translation of downstream target genes (22,23).( 3) CircRNAs function as protein scaffolds facilitating interactions between specific proteins (16,24).( 4) CircRNAs possess translational capacity, producing peptides that exert biological functions (25)(26)(27).(5) CircRNAs maintain the stability of mRNA and regulate its translation, facilitating or inhibiting the respective translation processes (22,28).(6) EIciRNAs and CiRNAs have been identified to be transcription regulators.EIciRNA locating in the nuclear can interact with the U1 small nuclear ribonucleoprotein (snRNP) and bind to RNA pol II, enhancing the transcription of their host genes.CiRNAs also modulate RNA pol II-mediated transcription, exerting a cisregulatory effect on upstream genes (29).CircRNAs can directly bind with nuclear transcription factors (TFs) to regulate their activity and function (30).Additionally, the transcription of some circRNAs might occur independently of host genes, regulated by TFs, as opposed to the general assumption of deriving from host transcript splicing (31).For instance, a set of circRNAs with transcription activation levels higher than those of host genes, identified as transcriptionally activated to a higher level than the host genes (TAH)-circRNAs, exhibited more TFs occupancy in their regulatory regions.Certain TFs, especially the validated super enhancer (SE) FOXA1 validated already, directly regulated the transcription of TAH-circRNAs (32,33).A novel genetic unit, the intragenic regulon (iRegulon), also differently regulates the expression of linear and circular RNA products, thereby manifesting distinct biological functions (30).To date, circRNAs have been established as participants in variety of physiopathological processes (14).

The biological functions and mechanisms of circ_0003945 in tumors 2.1 Biological functions of circ_0003945 in tumor cells
The expression of circ_0003945 was markedly elevated in tumor cell lines compared to the corresponding normal controls by quantitative real-time polymerase chain reaction (qRT-PCR) and statistical analysis of databases.Intriguingly, in gastric cancer and renal cell carcinoma cell lines, there was an aberrant downregulation of circ_0003945.The regulatory role of circ_0003945 in biological functions of tumor cells and potential mechanisms are as follows (Table 2).

Proliferation and cell cycle
Cancer cells exhibit distinct metabolic processes compared to normal cells, characterized by reduced oxidative phosphorylation or abnormal aerobic glycolysis, which drive their growth and proliferation (73).Employing techniques involving cell counting Kit-8 (CCK-8), ethylenediurea (EDU), or colony formation assay, researchers have uncovered that the overexpression of circ_0003945 enhanced proliferation across mostly tumors cell lines (Table 2).The MAPK pathway comprises four primary branches: ERK, JNK, p38/MAPK, and ERK5, each playing a crucial role in the cells proliferation, differentiation, migration, and invasion of cancer cells (74).Notably, the upregulation of circ_0003945 suppressed mircoRNA (miR)-1244 and activated MAP3K2 and MAPK key factors (including ERK, JNK, and p38), thereby augmenting the proliferation of prostate cancer cells (57).Circ_0003945 knockdown reduced the proliferation marker proliferating cell nuclear antigen (PCNA) by performing as an miR-638 sponge.This action effectively sequestered miR-638 from SZRD1, resulting in restrain of glioma cell proliferation (39).
Deviation in the cell cycle progression is fundamental to tumor cell proliferation.The proceeding of the cell cycle is regulated by metabolic enzymes and upstream regulators, primarily through cyclin-dependent kinases (CDKs) and other critical regulators like APC/C or SCF E3 ligase complexes (75).Knockdown of circ_0003945 could block the progression of lung adenocarcinoma cells from the G1 to S phase by regulating the binding of p27 to the cyclin-CDK complex.This intervention led to cell cycle to arrest at G0/G1 phase, consequently diminishing cell proliferation in vitro (51).This mirrored the function of circ_0003945 in glioma (62) and OC (68).Conversely, the upregulation of circ_0003945, acting as a tumor suppressor gene, The expression of circ_0003945 in human tumors.
Ovarian cancer Upregulated Oncogene OVCAR3, HO8910 Proliferation Migration (Continued) impeded cell cycle in the G1 phase in clear cell RCC cells (60).Ki-67 acts as specific biomarker of cellular proliferation, regulated through cell cycle-dependent transcription and protein degradation processes (76,77).Notably, overexpression of circ_0003945 was significantly associated with enhanced cellular of Ki-67 levels in NSCLC and HCC cells, implicating its roles in tumor cell proliferation (41, 43).In vivo, xenograft assays in mice had verified that stable knockdown of circ_0003945 led to reduced tumor volumes and weights compared matched controls.These findings indicate a promotive role for circ_0003945 in tumorigenesis, underlining its potential significance in cancer progression (Table 2).

Apoptosis
Apoptosis, an evolutionarily conserved mechanism, plays a critical role in cellular turnover and tissue regeneration.A hallmark of this process is the release of cytochrome c from mitochondria.The regulatory framework of apoptosis involves a self-amplifying cascade among pro-apoptotic and anti-apoptotic proteins of the Bcl-2 family, along with the initiator caspases (caspase-8, -9, and -10) and downstream effector caspases (caspase-3, -6, and -7).In tumor cells, a disruption of this balance often triggers the apoptotic pathway, highlighting its potential as a target in cancer therapies (78, 79).Knockdown of circ_0003945 increased pro-apoptotic proteins Bax and caspase-3 and decreased anti-apoptotic protein Bcl-2, thereby inducing apoptosis in CC cells.It might exert functions by serving as a ceRNA for miR−361−3p, hampering SOX4 and thus impeding tumor cell progression (56).Knockdown of circ_0003945 reduced the expression of Bcl-2, conversely while elevating Bax and caspase-3 in OC cells.And overexpressing miR-382-5p could reverse this impact on apoptosis-related proteins and downstream PRPF8 gene (68).Furthermore, the study reported that, in NSCLC cell, silencing of circ_0003945 led to the downregulation of apoptosis-associated genes and proteins such as c-IAP1, Bcl-2, Survivin, and cell cycle protein CDK6, cyclin D1 were, while upregulation p27 and Bax (51).C-MYC is a multifunctional TF often associated with hepatocarcinogenesis. Its overexpression can enhance hepatocyte apoptosis (80).Previous studies indicated c-MYC as a crucial driver in transforming hepatocytes from the G0/G1 phase to the S phase (81).Overexpressing circ_0003945 notably increased the expression of c-MYC and cellular DNA synthesis, inhibited apoptosis in HCC.This mechanism accompanied by sponging miR-1294 (66).

Autophagy
Autophagy is an essential catabolic process that recycles limited intracellular resources and mediates the degradation of damaged or redundant organelles under stress conditions to preserve cellular functionality (73).Previous studies have shown that epithelial cells can evade anoikis though autophagy and epithelial-mesenchymal transition (EMT), facilitating cell migration and invasion (82,83).The accumulation of LC3B-II and the conversion from LC3B-I to LC3B-II is sensitive autophagy induction markers.In CRC, silencing circ_0003945 decreased total autophagosomes and autolysosomes formation.This knockdown impeded the expression of LC3B-II, lowered LC3B-I/II conversion rates, and decreased the degradation of key autophagy-related proteins like Beclin1, ATG7, and FOXO1.It was demonstrated that modulating circ_0003945 affected the miR-582-5p/FOXO1 axis, thereby inducing autophagy and promoting migration and invasion of CRC cells (40).

Metastasis
The invasion-metastasis cascade, pivotal in cancer progression, involves extracellular matrix (ECM) degradation by a broad extent of cells and matrikines such as MMPs, Versican, and others.MMP-9, in particular, remodels the ECM, influencing tumor invasion, metastasis, and angiogenesis.The alteration of tumor stroma and release of angiogenic factors are key strategies in this process, supported by MMP-mediated vasculature growth (84)(85)(86).EMT is a cellular-transformed process marking by changes from Ecadherin-expressing epithelial cells to a mesenchymal phenotype expressing vimentin and N-cadherin, endowing tumor cells to migrate and invade (83,87).In HCC cells, downregulating circ_0003945 enhanced E-cadherin and reduced N-cadherin and a-SMA levels, suggesting it EMT and invasion (52).Overexpression of circ_0003945 in NSCLC cells increased MMP9 and Fibronectin, reduced E-Cadherin via KLF4 and miR-3182, and activated Rac1/

Angiogenesis
Tumor cells often secrete high levels of pro-angiogenic factors which contribute to the development of an abnormal vascular network.However, the immaturity of tumor blood vessels impairs their functionality for the tumor microenvironment (TME) and increases risk of metastatic dissemination (89).Among these factors, VEGF, particularly VEGFA from the VEGF family, plays a pivotal role.VEGFA is crucial for cell proliferation, invasion, and angiogenesis in various malignancies (90).Circ_0003945 sponged miR-199a to upregulate VEGFA to promote CRC progression.And inhibiting miR-199a or overexpressing VEGFA could reverse the tumor-suppressing effects of circ_0003945 knockdown (54).Meanwhile, the tube formation assay in glioma cell lines revealed that knockdown of circ_0003945 led to reduced angiogenesis, evidenced by a decrease in branch formation.This suggested that circ_0003945 influenced the angiogenic capacity of glioma cells by the miR-638/SZRD1 axis (39).

Immune escape
Malignant tumor cells often evade the surveillance of immune system, where immune cells can normally identify and eliminate malignancies.Tumor cells alter their structure, effect genes and protein expression within TME to evade immune surveillance, presenting a significant challenge to immunotherapy (91,92).Cancer-associated fibroblasts (CAFs), prevalent in the TME, can contribute to this evasion.They aid tumor progression through ECM remodeling, growth factor production, cytokines and chemokines secretion, and metabolic and angiogenic modulation (93).In hepatitis virus-induced cirrhosis or liver cancer, CAFs originated from hepatic stellate cells and gradually transformed into a major ECM source due to the stroma cell accumulation.HCC-associated fibroblast implied CAF-derived CXCL11 enhanced cell invasion by regulating the circ_0003945/miR-4756/IFIT1/3 axis.Tumor cell morphology was altered from flattened to spindle-shaped and produced more pseudopods, causing elevated cell proliferation, DNA synthesis, migration ability, and protein levels of migration-related markers vimentin and twist (67).In pancreatic adenocarcinoma (PAAD), the circ_0003945/miR-494 axis regulated PAAD progression by CXCR4 and ZEB1, key mediators of tumor immune cell infiltration.Elevated CXCR4 and ZEB1 levels associated with immune cell markers and immune checkpoint proteins.This axis promotes M2 macrophage polarization of tumor-associated macrophages (TAMs), Treg recruitment and activation in TME, and induced T-cell depletion for immunological escape (53).

The potential molecular mechanisms of circ_0003945 regulating tumorigenesis
CircRNAs exert multiple biological functions, notably though the ceRNA mechanism mentioned upward.Previously described as the "Rosetta stone of a hidden RNA language," ceRNA has garnered significant attention and undergone extensive research, bolstered by next-generation sequencing technology (94).CeRNAs cross regulate each other through sequestration of shared miRNAs and form complex regulatory networks based on their miRNA signature (95).Advances in sequencing technology have increasingly shown that ncRNAs, such as pseudogenes, small and long ncRNAs, and circRNAs, are key in biological processes and tumorigenesis (96,97).MiRNAs are a short, single-stranded, and highly conserved class of 18-to 24-nt endogenous ncRNAs, undergo a multistep biogenesis process.Initiated by RNA pol II, the process produces primary-microRNAs (pri-miRNAs), which are cleaved into precursor-miRNA (pre-miRNA) by the Drosha-DGCR8 complex (98).Exported to the cytoplasm, these pre-miRNAs are further processed by ribonuclease Dicer into small double-stranded RNA (dsRNA) fragments.The functional strand is incorporated into the Argonaute (AGO) protein, forming the RNA-induced silencing complex (RISC).This complex functions as the primary effector in biological processes (99, 100).Mature miRNAs bind complementarily to the 3′untranslated region (3′UTR) of the target mRNA through miRNA response elements, either by base pairing or via additional sequence elements.AGO interacts with the polyA-tailed binding in the 3′end of mRNA by recruiting adapter protein TNRC6.This interaction facilitates post-transcriptional mRNA degradation and translation repression (101)(102)(103).
The mechanism of ceRNA involves endogenous transcripts, rich in miRNA-binding sites, that can remarkably dimmish the miRNAmediated repression of target gene mRNA.CircRNAs, acting as sponges, compete with these miRNAs to sequester them from their original targets (97, 104-106).This forms a complex ceRNA network, where transcripts compete for miRNA, collaboratively modulating miRNA activity (107).Circ_0003945 networks link to the function of protein-coding mRNAs and related signaling pathways, demonstrating the extensive influence of these interactions (Table 2).

The clinical significance and prognostic value of circ_0003945 in tumors
Overexpression of circ_0003945 tended to mean more aggressive clinicopathological parameters and poorer prognosis for tumor patients in major conditions.Its upregulation was more prone to increased therapy resistance through multiple mechanisms.

The clinical parameters of circ_0003945 in tumors
Numerous studies have identified circRNAs as potential diagnostic biomarker in cancers (12).Specifically, circ_0003945 expression was found to be higher in various cancer tissues compared to paired adjacent noncancerous tissues, though it is notably decreased in gastric cancer and renal cell carcinoma.(Table 2).High circ_0003945 levels correlated with aggressive clinicopathological characteristics such as invasive histological types, poor differentiation, higher recurrence rates, extensive microvascular invasion, and advanced tumor-nodemetastasis (TNM) stages, including larger tumor size, more lymph node and distant metastases (Table 3).Kaplan-Meier survival analysis showed that patients with high circ_0003945 levels had shorter overall survival (OS), indicating a poorer prognosis (Table 3).Moreover, higher circ_0003945 expression was linked to reduced recurrence-free survival (RFS) and is an independent predictor of time to recurrence (TTR) in hepatocellular carcinoma.In tamoxifen-resistant patients, elevated circ_0003945 levels were associated with worse distant metastasis-free survival (DMFS) (65).
Currently, there are no related clinical trials targeting circ_0003945 in human diseases.However, some other clinical trials targeting circRNAs have been carried out so far.For example, studies identified and compared differentially expressed of relevant circRNAs in various cells or tissues.Independent cohorts confirmed FIGURE 4 Molecular mechanisms of circ_0003945 in different diseases.

Circ_0003945-related therapeutic resistance
Cancer therapeutic resistance is broadly classified into intrinsic and acquired (including adaptive) types, with tumor cells traditionally categorized as either drug-sensitive or drug-resistant.Resistance often involves pathway-based mechanisms, with activation of drug-inhibited effector proteins upstream, parallel, or downstream of the primary targets (115).The upregulation of circ_0003945 has been linked to augmented cisplatin resistance in TNBC cells.Circ_0003945 curbed TNBC sensitivity to cisplatin through the miR-300/ASF1B axis by activating PI3K/AKT/mTOR pathway (64).Knockdown of circ_0003945 hampered SEMA6D to reversing cisplatin resistance via sponging miR-506-3p by restraining the Wnt/b-catenin signaling pathway in osteosarcoma (70).Conversely, as a tumor suppressor gene in gastric cancer, circ_0003945 hindered cisplatin resistance through the miR-300/ KAT6B axis (59).Additionally, the depletion of circ_0003496 suppressed tumor growth and enhanced doxorubicin (DXR) sensitivity in osteosarcoma by targeting KLF12 via miR-370 (37).In NSCLC, its downregulation significantly mitigated resistance to docetaxel, DXR, and gefitinib, via restricting KLF4 through modulation of miR-3182 (43).Furthermore, research discovered a potential mechanism in tamoxifen resistant, where circ_0003945 acting though miR-1296-5p/STAT1 axis, contributed to tamoxifenresistant in luminal breast tumors (65).In glioma cells, inhibiting circ_0003945 increased radiosensitivity by weakening RNF2 and counteracting the effects of miR-433-3p (61).
Generally, the upregulation of circ_0003945 was consistently associated with therapeutic resistance by signaling pathways in drug-resistant cancer cell lines and tissues (37,43,59,61,64,65,70).It might provide novel perspective on reserving drug resistance or improving radiosensitivity in tumor therapy.

Role of circ_0003945 in non-malignant conditions
In cardiovascular disease, circ_0003945 was correlated with microcirculatory perfusion.It inhibited the migratory activity and promoted M2 polarization in macrophages, declining the productions of cytokines TNF-a, interleukin (IL)-1b, and MCP-1 and the PI3K/AKT/mTOR pathway.
The expression of circ_0003945 might predict prognosis in extracorporeal membrane oxygenation (ECMO) patients with cardiogenic shock (45).In a canine ECMO model, the modifications of pulsatility improved microcirculatory perfusion and endothelial integrity.The upregulation of circ_0003945 stabilized endothelial tight junction markers ZO-1 and occluding.It followed by modulating the eNOS activity and inhibiting the NF-kB signaling pathway, pivotal in this protective mechanism (44).Furthermore, circ_0003945 was upregulated in DR, when knocked down, it alleviated high glucose-triggered oxidative stress and dysfunction in human retinal microvascular endothelial cells (hRMECs).It might offer a promising therapeutic target through the miR589-5p/EGR1 axis for DR (35).In OA, exosome-mediated circ_0003945 participated in IL-1b-induced chondrocyte damage.It stimulated WNT5B via hindered miR-149-5p, affecting chondrocyte proliferation, apoptosis, migration, invasion, inflammation, and ECM degradation (46).Circ_0003945 was found to be downregulated in placental tissues from patients with PE compared to healthy controls.Its knockdown impeded trophoblast cell proliferation and migration via miR-1244/ FOXM1 axis (47).Interestingly, circ_0003945 was significantly upregulated in cow mammary gland tissue, influencing milk fat metabolism by sponging miR-331-3p (48).These studies underscore the potential role of circ_0003945 in various diseases beyond tumors.

Conclusions and prospects
CircRNAs have garnered widespread attention in contemporary medical research due to their indispensable roles in human diseases pathogenesis.In this review, we discussed a compendium of studies that document the aberrant expression of circ_0003945 in various human diseases.These studies elucidate the specific cellular and biological functions of this circRNA.In most malignancy cases, circ_0003945 served as an oncogene.It functioned as a miRNA sponge, inhibiting the transcription of miRNAs, and thereby activates downstream effector genes.However, in a minority of malignancies, such as GC and RCC, circ_0003945 acted as a tumor suppressor gene.Research has indicated that circ_0003945 could alter the biological behaviors of tumor cells by modulating signaling pathways such as MAPK, Wnt/b-catenin, PI3K/AKT/mTOR, Rac-FAK1, and others.The expression levels of circ_0003945 were strongly correlated with the clinicopathological features of patients, and survival analyses also provided valuable insights into prognosticating clinical outcomes.Moreover, circ_0003945 affected sensitivity to chemotherapy and radiotherapy through alterations in its molecular mechanisms.This finding indicated that circ_0003945 might serve as a valuable predictive biomarker for clinical management of diseases.
Nonetheless, current research on circ_0003945 remands relatively restricted.The primary issue is the obscurity surrounding the upstream regulatory mechanisms of circ_0003945.Within the TRCirc database, the TF-peak of SE FOXA1 is located squarely in the transcriptional domain of circ_0003945.Further empirical validation is needed to determine whether a specific TF can directly and independently regulate the transcription of circ_0003945, thereby impacting the function of downstream proteins.Additionally, most molecular mechanisms identified for circ_0003945 are limited to the model of ceRNA, leaving other functionalities of circRNAs yet to be elucidated.Moreover, there is an insufficient exploration of the correlations with unmentioned sub-histological even pathological types of tumors in experiments.Furthermore, significant advancements in clinical trials, including consecutive biopsies of tumor tissues and circulating plasma sampling, are essential to definitively ascertain whether circ_0003945 can serve as a reliable clinical diagnostic maker.Finally, when relevant clinical trials concerning circ_0003945 are conducted and yield valid conclusions, the potential of circ_0003945 as a target for clinical therapy is expected to be realized.

TABLE 1
Alias of circ_0003945 and different splicing methods from UBAP2 gene in diseases.

TABLE 3
Clinicopathological parameters of circ_0003945 in various cancers.