Oncogenic hijacking of a conserved hsa_circ_0005140/miR-762/NFIX axis drives retinoblastoma proliferation through context-dependent activation

Oncogenic hijacking of a conserved hsa_circ_0005140/miR-762/NFIX axis drives retinoblastoma proliferation through context-dependent activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Oncogenic hijacking of a conserved hsa_circ_0005140/miR-762/NFIX axis drives retinoblastoma proliferation through context-dependent activation Jihan Luo, Hao Yao, Xiyuan Zhou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8392296/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Circular RNAs play pivotal roles in cellular regulation, yet their mechanisms in retinal cells remain incompletely understood. Through comparative analysis of Y79 retinoblastoma and ARPE-19 retinal pigment epithelial cells, hsa_circ_0005140 was identified as a functionally active circRNA engaging miR-762 and nuclear factor I X (NFIX). Methods: Comprehensive functional assays included: RT-qPCR validation; Dual-luciferase reporter assays confirming molecular interactions; Phenotypic characterization (CCK-8/EdU/Transwell/Flow cytometry); Western blotting and xenograft models for in vivo validation‌. Results: Differential expression, hsa_circ_0005140 showed 596-fold higher expression in Y79 than ARPE-19 (p<0.001), yet exhibited conserved regulatory functions in both cell types. Functional axis, acts as miR-762 sponge to upregulate NFIX (60% luciferase activity reduction, p<0.001). Proliferative effects, modulated cell cycle progression and apoptosis in both Y79 (p<0.01) and ARPE-19 (p<0.05). ‌Downstream effects, hsa_circ_0005140 overexpression led to elevated IL-6/IL-8 levels (2.1-3.5 fold changes) and context-dependent TNF-α responses. In Vivo Validation‌, Xenografts showed 2.3-fold tumor growth promotion by hsa_circ_0005140 (p<0.01), reversible by miR-762 overexpression.‌ ‌Conclusions: This study establishes that ‌aberrant hsa_circ_0005140 overexpression activates a fundamental regulatory axis‌(circRNA/miR-762/NFIX) which exerts proliferative effects in both pathological and physiological contexts. The observed inflammatory marker dysregulation suggests broader functional consequences of circRNA dysregulation in retinal cells. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Molecular biology circular RNA sponge Hsa_circ_0005140 miR-762 NFIX Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Circular RNAs (circRNAs) represent a novel class of noncoding RNAs that regulate cellular activities. Characterized by their closed circular structure, circRNAs lack a 5' cap and 3' poly(A) tail structures, are predominantly found in the cytoplasm or exosomes, and are resistant to RNA exonucleases [ 1 ] . Their stability and resistance to degradation make them promising for various applications. Recent studies have highlighted the involvement of circRNAs in numerous biological processes, including cell growth, the stress response, and disease progression. They show considerable potential as diagnostic markers and therapeutic targets in cancers or other diseases, influencing processes such as cell viability, proliferation, apoptosis, autophagy, migration, and invasion. CircRNAs are generally generated through postsplicing of precursor microRNA (miRNA) exons [ 2 , 3 ] . On the basis of their original sequences, circRNAs are classified into three types: exon-intron, intron-intron, and exon-intron (EIcircRNA) types [ 4 ] . The recognized biological functions of circRNAs include the following [ 5 ] : miRNA sponging, protein binding regulation, gene transcription regulation and encoding function. The most significant function of circRNAs is their role as miRNA sponges [ 6 ] . CircRNAs with miRNA binding sites directly bind to miRNAs, inhibiting their function and regulating target gene expression through what is known as the circRNA ceRNA axis. This multifactorial interaction network suggests that targeting specific circRNAs could alter the levels of multiple disease-related RNAs simultaneously, making circRNAs valuable in the study of diagnosis and therapeutic targets for complex diseases [ 7 ] . In retinal cells, circRNAs are increasingly recognized as modulators of proliferation and inflammatory responses, yet their conserved versus cell-type-specific functions remain poorly characterized. In retinal physiology and pathology, circRNAs play pivotal roles through miRNA sponging and protein scaffolding mechanisms. They are implicated in several retinal disorders [ 8 ] , including diabetic retinopathy (DR), glaucoma, proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD) [ 9 ] , and ocular cancers such like retinoblastoma (RB) [ 10 ] . In DR, circRNAs like circ_0005015 [ 11 ] and circHIPK3 [ 12 ] regulate vascular dysfunction by sponging specific miRNAs (e.g., miR-519d-3p and miR-30a-3p) to modulate endothelial cell proliferation and angiogenesis. Similarly, cPWWP2A [ 13 ] and cZNF532 [ 14 ] maintain vascular integrity by sequestering miR-579 and miR-29a-3p, respectively. In glaucoma, cZNF609 exhibits neuroprotective effects by derepressing miR-615 targets such as METRN, thereby mitigating retinal ganglion cell degeneration [ 15 , 16 ] . PVR involves circ_0043144-driven ARPE-19 cell proliferation and cytokine secretion (CCL2, VEGF-A), contributing to epiretinal membrane formation [ 17 ] . AMD pathogenesis is linked to circNR3C1 downregulation, which normally protects RPE cells via the miR-382-5p/PTEN/AKT/mTOR pathway against oxidative stress [ 18 ] . Three lncRNAs (ENSGALT00000098661, ENSGALT00000100816, and MSTRG.16980.1) and one circRNA (novel_circ_010168) in the ncRNA-mRNA regulatory network were identified as key molecules influencing circadian rhythm by regulating AOX1 in retinal metabolism [ 19 ] . Research on RB-related circRNAs has identified several oncogenic circRNAs, such as circ-FAM158A [ 20 – 25 ] , circ-DHDDS [ 26 – 29 ] , circ-E2F3 [ 30 – 33 ] , circ-TRHDE [ 34 ] , circ-E2F5 [ 35 ] , circ-ODC1 [ 36 ] , circ-RNF20 [ 37 ] , and circ-0007534 [ 38 ] , which promote disease progression and metastasis. Conversely, circ-TET1 [ 39 – 40 ] , circ-SHPRH [ 41 ] , circ-MKLN1 [ 42 ] , and circ-CUL2 [ 43 ] act as tumor suppressors in RB. In this study, we analyzed high-throughput sequencing data from Y79 retinoblastoma cells and ARPE-19 retinal pigment epithelial cells, and identified hsa_circ_0005140 as being markedly overexpressed in RB. We had designed a series of studies to explore its mechanism of action in RB. We confirmed the existence of the molecular regulatory axis of hsa_circ_0005140 /miR-762/NFIX. At the same time, cell function experiments revealed an unexpected phenomenon - the pro-proliferative function of this axis is not specific to RB tumor cells, but also exists in ARPE-19 cells. Through the analysis of these experimental data, we speculate that RB tumor cells may regulate a fundamental regulatory pathway crucial for retinal cell proliferation by abnormally upregulating hsa_circ_0005140, thereby driving their malignant proliferation. 2. Materials and Methods 2.1. Cell culture The retinoblastoma cell line Y79 (ATCC HTB-18) RRID: CVCL_1895 and the normal retinal pigment epithelial cell line ARPE-19 (ATCC CRL-2302) RRID: CVCL_0145 were procured from Cellverse Co., Ltd. (Shanghai, China). Both cell lines were maintained in RPMI-1640 medium (Gibco, Los Angeles, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Opcel Biotechnology Co., Ltd., Inner Mongolia, China). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 . 2.2. High-throughput sequencing High-throughput sequencing was performed on Y79 and ARPE-19 cells by GENESEED Biotechnology Co., Ltd. (Guangzhou, China). This sequencing aimed to generate circRNA expression profiles for retinoblastoma, identify the top 10 differentially expressed (upregulated and downregulated) circRNAs, and explore their downstream interaction networks. The target genes were also determined for further research. The raw sequence data used for analysis are available in NCBI under the Sequence Read Archive (SRA), with the accession number SRP658189. 2.3. Real-time quantitative polymerase chain reaction (RT-qPCR) RNA was extracted from Y79 and ARPE-19 cells via TRIzol reagent (Biosharp, Beijing, China). Complementary DNA (cDNA) was synthesized via a reverse transcription kit(with dsDNase) from Biosharp. RT-qPCR was conducted using Universal SYBR qPCR Master Mix (Biosharp). The RNA expression levels were quantified via the 2 − ΔΔCt method, with GAPDH or U6 (for miR-762) serving as internal controls. Each sample was analyzed in triplicate. The following primers were used: circ_117102-F1: 5'-CGGTGGATCAATGGAACTGG-3', circ_117102-R1: 5'-ACTAAAATCACCAGCAGAGCA-3' circ_5140-F2: 5'-CACATGCGGGCACACTCA-3', circ_5140-R2: 5'-TTTCCGAGGCGTTATCTCCC-3' circ_54598-F2: 5'-CAAGCAGAGATAGAGAGCATAGT-3', circ_54598-R2: 5'-CTCAGATGCAGCAGGAAGA G-3' circ_60927-F1: 5'-GACATCCAGGCCACAGACAA-3', circ_60927-R1: 5'-CCAGTCTTCCCCTTCCCTGA-3' circ_132246-F1: 5'-CCCAGCTTAGTCAAACAGAACAA-3', circ_132246-R1: 5'-CTGTCTTGTTCGCTCCTCCA-3' NFIX-F: 5'-GCCTTGACTCCTCCATCACC-3', NFIX-R: 5'-CAGGACTGAGACTGCTGTGG-3' miR-762 RT: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTCGG, miR-762-F: 5'-GGGGCTGGGGCCGGGG-3', miR-762-R: 5'-AGTGCAGGGTCCGAGGTATT-3' miR-762 mimics: 5'-GGGGCUGGGGCCGGGGCCGAGC-3' mimics NC: 5'-UCCUGUACUGAGCUGCCCCGAG-3' miR-762 inhibitor: 5'-GCUCGGCCCCGGCCCCAGCCCC-3' inhibitor NC: 5'-UCUACUCUUUCUAGGAGGUUGUGA-3' GAPDH-F: 5'-AGAAGGCTGGGGCTCATTTG-3', GAPDH-R: 5'-GCAGGAGGCATTGCTGATGAT-3' U6-F: 5'-CTCGCTTCGGCAGCACA-3', U6-R: 5'-AACGCTTCACGAATTTGCGT-3' 2.4. Construction of overexpression and interference plasmids The full-length sequence (1555 bp) of hsa_circ_0005140 was amplified from cDNA via PCR and subsequently cloned and inserted into the pLC5 ciR vector via an In-Fusion one-step cloning kit (Yeasen, Shanghai, China). The sequences of primers used for amplification were as follows: C5140-F: 5'-CATTATTTTTCTCTCTCTCGGGACAGCCGCCTGCT-3' C5140-R: 5'-AGTATGGAGTTGTTAGCTAGGATCCAGTTGTTCTTACCTGAGTGTGCCCGCATGTGGG-3' To construct the interference plasmid, the shRNA sequences targeting 5140-Sh GGGCACACTCAGGGGGGGACA were as follows: F: GATCCGGGCACACTCAGGGACATCAAGAGTCCCCCTGTGTGCCTTTTTG R: AATTCAAAAAAGGGCACACTCAGGGGGACACTCTTGATGTCCCCCTGAGTGTGCCCG The BamHI and EcoRI restriction sites were included at the 5' and 3' ends, respectively. The annealed double-stranded primers were ligated into the pLVX-shRNA2 vector. Plasmid extraction was performed via an endotoxin-free plasmid extraction kit (Magen, Guangzhou, China), and transfections were conducted with Lipo8000™ Transfection Reagent (Beyotime, Shanghai, China). 2.5. Cell transfection and selection of stably transfected cell lines 293T cells were used to produce viral particles containing the overexpression plasmid hsa_circ_0005140-pLC5-ciR and interference plasmid hsa_circ_0005140-pLVX-shRNA2, along with copackaging plasmids (psPAX.2, pMD2.G). The viral supernatant was collected, concentrated, and stored at -80°C. Target cells (5 × 10 5 cells/well) were seeded into 6-well plates and incubated at 37°C with 5% CO 2 . After 48 hours of viral infection, the infection efficiency was assessed via fluorescence microscopy. Stably transfected cell lines were selected with 2 µg/mL puromycin for 2–3 weeks. 2.6. Cell counting Kit-8 (CCK-8) assay To assess cell proliferation, a CCK-8 assay was performed via a CCK-8 kit (SparkJade, Shandong, China). Approximately 5×10³ cells per well were seeded into 96-well plates and allowed to adhere for 3 hours. At days 1, 2, 3, 4, and 5, 10 µL of CCK-8 solution was added to the wells. The cells were then incubated in a 5% CO₂ environment at 37°C for 30 minutes. The absorbance was measured at 450 nm via a microplate reader. 2.7. EdU assay Cell proliferation was further evaluated via an EdU-594 Cell Proliferation Detection Kit (Labgic, Beijing, China). After EdU labeling for 6 hours, the cells were washed with phosphate buffered saline (PBS), fixed, and permeabilized. The cells subsequently underwent a click reaction, followed by Hoechst 33342 staining. The samples were then visualized under a fluorescence microscope, and images were captured. 2.8. Transwell analysis Cell migration and invasion were analyzed via 24-well transwell chambers (Corning, NY, USA). After transfection, Y79 and ARPE-19 cells were placed in the upper chambers, while the lower chambers were filled with RPMI-1640 medium supplemented with 10% FBS. After 24 hours of incubation, the cells were fixed in methanol and stained with 0.1% crystal violet. The migrated cells were counted in five random fields under a microscope at 100× magnification. For invasion assays, transwell inserts were precoated with Matrigel (Corning). 2.9. Flow cytometry Forty-eight hours posttransfection, Y79 and ARPE-19 cells were collected, trypsinized, and washed with PBS. The cells were fixed in 80% ethanol for 2 hours, treated with RNase A, and stained with propidium iodide (PI). The cell cycle distribution was determined via FACScan flow cytometry. Apoptotic cells were measured via the Annexin V-PE/7-AAD apoptosis detection kit (KeyGen) according to the manufacturer's guidelines, and data were acquired via FACScan flow cytometry. 2.10. Dual-luciferase reporter assay The circ_0005140 sequence, containing either wild-type or mutant miR-762 binding sites, was cloned and inserted into the psiCHECK2 vector (Promega, Beijing, China) to generate circ_0005140 wt or circ_0005140 mut constructs. Similarly, NFIX 3'UTR sequences with wild-type or mutant binding sites were cloned and inserted into the same vector. Y79 cells were cotransfected with the constructed reporters and miR-762 or miR-NC. Luciferase activity was measured via the Dual-Lucy assay kit (Labic). 2.11. circRNA cellular function rescue assay Functional validation of the circ_0005140/miR-762/NFIX axis was performed through CCK-8 assays, EdU assays, transwell analysis, flow cytometry, and RT-qPCR, utilizing overexpression and knockdown of miR-762 (Tsingke Biotechnology Co., Ltd., Beijing, China). 2.12. Western blotting assay (WB) Y79 and ARPE-19 cells were lysed in RIPA buffer (Biosharp) to extract total protein, which was quantified via a BCA protein assay kit (SparkJade). Proteins (30 µg) were subjected to SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked overnight with the indicated antibodies against NFIX, TNF-α, IL-6, IL-8, or GAPDH and then incubated with HRP-conjugated secondary antibodies (Proteintech Group, Rosemont, IL, USA) for 2 hours. Detection was conducted via the use of enhanced chemiluminescence (ECL) reagent (FUDEBio, Hangzhou, China). 2.13. Xenograft assay The xenograft assay, which was approved by the Animal Ethics Committee of Chongqing Medical University (IACUC-SAHCQMU-2024-00099), followed National Institutes of Health guidelines and the study was reported in accordance with ARRIVE guidelines. At the initiation of the experiment, the BALB/c nude mice were 5 weeks old with an average body weight of approximately 15 g. Y79 cells (1×10⁷) stably expressing various constructs (Y79-OE-NC, Y79-C5140-OE, Y79-C5140- OE + miR-762 mimics, Y79-SH-NC, Y79-C5140-SH, Y79-C5140-SH + miR-762 inhibitor) were injected subcutaneously into the right flanks of 5-week-old BALB/c nude mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd) (n = 5 per group). The tumor volume was recorded every two days. After three weeks, the mice were sacrificed, and the tumors were excised for weight measurement and analysis of the miR-762 and NFIX levels. Ki-67 and NFIX were also measured by immunohistochemical staining. The euthanasia method for BALB/c nude mice was performed as follows: the posterior part of the mouse's head was firmly grasped with the thumb and index finger of the left hand, followed by a downward pressure. Simultaneously, the mouse's tail was held with the right hand and pulled forcefully backward and upward to induce cervical dislocation, resulting in instantaneous death of the mouse. 2.14. Statistical analysis The data are presented as the means ± standard deviations. Statistical significance was determined via Student’s t test for two-group comparisons or two-way ANOVA for multiple groups, implemented in GraphPad Prism 9 (GraphPad, San Diego, CA, USA). P < 0.05 was considered statistically significant. All experiments were conducted at least in triplicate. 3. Results 3.1 Differential expression of circular RNA in Y79 cells and ARPE-19 cells. To obtain the circRNA expression profile in retinoblastoma cells, we conducted high-throughput sequencing of Y79 and ARPE-19 cells, with ARPE-19 cells used as a control (Fig. 1 A). We identified the top 10 circRNAs with significant differential expression (upregulated and downregulated) between Y79 and ARPE-19 cells (Table 1 ). Notably, we focused on hsa_circ_0005140, the second most highly expressed circRNA, which has an appropriate molecular weight and binding site availability. The downstream interaction network involving miR-762 and NFIX (Fig. 1 B), which was predicted via high-throughput sequencing, also has clinical relevance, as this signaling pathway has been validated in study of acute lung injury [ 44 ] . Consequently, we selected hsa_circ_0005140 as the target gene for further investigation into the role of the miR-762/NFIX signaling pathway in the occurrence and progression of retinoblastoma. We then utilized RT-qPCR to measure the levels of miR-762 and NFIX in both Y79 and ARPE-19 cells. The results indicated that miR-762 levels were significantly lower in Y79 cells than in ARPE-19 cells, whereas NFIX levels were significantly higher in Y79 cells (Fig. 1 C, 1 D). Additionally, we verified the expression levels of the top three overexpressed circRNAs (hsa_circ_0117102, hsa_circ_0005140, and hsa_circ_0054598) and the top two underexpressed circRNAs (hsa_circ_0132246 and hsa_circ_0060927) in Y79 and ARPE-19 cells via RT-qPCR, which confirmed the high-throughput sequencing results (Fig. 1 E). 3.2 Hsa_circ_0005140 knockdown suppressed Y79 and ARPE-19 cell proliferation, migration and invasion and facilitated apoptosis. miR-762 suppression eliminated the effects of hsa_circ_0005140 knockdown on Y79 and ARPE-19 cells. We subsequently performed functional assays by transfecting 293T cells with either the overexpression plasmid hsa_circ_0005140-pLC5-ciR or the knockdown plasmid hsa_circ_0005140-pLVX-shRNA2, along with the packaging plasmids psPAX.2 and pMD2. G, to produce viral solutions. These viral solutions were transfected into Y79 and ARPE-19 cells, from which we obtained stable cell lines through puromycin selection. Next, we validated the role of hsa_circ_0005140 and miR-762 in retinoblastoma through functional cell assays, including CCK-8, EdU, transwell migration, and invasion experiments, and the results demonstrated that the overexpression of hsa_circ_0005140 increased the proliferation, migration, and invasion of Y79 and ARPE-19 cells, whereas the knockdown of hsa_circ_0005140 inhibited these processes (Fig. 2 A, 2 B, 2 C, 2 D, 2 E). The overexpression of miR-762 counteracted the proliferation, migration, and invasion-promoting effects of hsa_circ_0005140 in Y79 and ARPE-19 cells (Fig. 3 A, 3 B, 3 C, 3 D, 3 E). Conversely, knockdown of miR-762 reversed the inhibition of these processes caused by hsa_circ_0005140 knockdown. Flow cytometry analysis of apoptosis and the cell cycle revealed that overexpression of hsa_circ_0005140 reduced apoptosis and decreased G0/G1 phase arrest in both cell lines, while knockdown promoted apoptosis and enhanced G0/G1 phase arrest (Fig. 4 A, 4 B, 4 C, 4 D); miR-762 overexpression negated the inhibitory effects of hsa_circ_0005140 overexpression on apoptosis and G0/G1 phase arrest in both cell lines. Similarly, knockdown of hsa_circ_0005140 enhanced apoptosis and G0/G1 phase arrest, an effect that was reversed by miR-762 knockdown (Fig. 4 E, 4 F, 4 G, 4 H). These findings suggest that hsa_circ_0005140 may negatively regulate miR-762 by directly targeting it. 3.3 hsa_circ_0005140 interacts with hsa-miR-762, and hsa-miR-762 interacts with the NFIX gene. A dual-luciferase reporter assay was used to validate the circ_0005140/miR-762/NFIX signaling pathway. Compared with NC transfection, transfection of the wild-type hsa_circ_0005140 reporter gene plasmid with miR-762 mimics significantly reduced luciferase activity. In contrast, no significant difference was observed in the mutant hsa_circ_0005140 plasmid group. These results suggest an interaction between hsa_circ_0005140 and miR-762 (Fig. 5 A). Compared with the NC, the overexpression of miR-762 mimics significantly decreased the luciferase activity of the wild-type NFIX 3'UTR reporter gene plasmid. However, there was no significant reduction in luciferase activity in the mutant NFIX 3'UTR group. These findings indicate a potential interaction between NFIX and miR-762 (Fig. 5 B). 3.4 Hsa_circ_0005140 knockdown suppressed RB tumor growth in vivo. The effect of hsa_circ_0005140 on RB progression in vivo was investigated via xenotransplantation assay. The excised tumors are displayed in Fig. 8A and 8B, and the tumor growth rates and weights were recorded. The overexpression of hsa_circ_0005140 significantly promoted tumor growth, with the tumor weight in the overexpression group exceeding that in the control group. Conversely, overexpression of miR-762 attenuated this effect (Fig. 6 A). In contrast, knockdown of hsa_circ_0005140 notably inhibited tumor growth, resulting in significantly lower tumor weights than those in the control group. Additionally, knockdown of miR-762 mitigated the growth inhibition associated with hsa_circ_0005140 knockdown (Fig. 6 B). Furthermore, hsa_circ_0005140 overexpression led to increased NFIX expression in tumor tissue, whereas miR-762 overexpression significantly reversed this effect. Conversely, hsa_circ_0005140 knockdown reduced NFIX expression, which was reversed by miR-762 knockdown. Notably, hsa_circ_0005140 overexpression and knockdown did not significantly affect miR-762 levels; however, miR-762 overexpression significantly increased its expression, whereas knockdown decreased it (Fig. 6 C). 3.5 Western blotting analysis WB was employed to assess alterations in NFIX levels within the hsa_circ_0005140/miR-762/NFIX signaling pathway, along with variations in the levels of related inflammatory cytokines, including IL-6, IL-8, and TNF-α(Figure 7 A, 7 B). The overexpression of hsa_circ_0005140 significantly increased the protein levels of NFIX, IL-6, and IL-8 in both Y79 and ARPE-19 cells, whereas the overexpression of miR-762 reversed these effects. Conversely, knockdown of hsa_circ_0005140 markedly reduced the expression of NFIX, IL-6, IL-8, and TNF-α in both cell lines, with miR-762 knockdown reversing these reductions. Notably, neither the overexpression of hsa_circ_0005140 nor the expression of miR-762 significantly affected TNF-α levels in Y79 cells; however, hsa_circ_0005140 knockdown significantly decreased TNF-α, which was reversed by miR-762 knockdown. While hsa_circ_0005140 knockdown significantly reduced IL-6 levels in ARPE-19 cells, miR-762 knockdown mitigated this decrease, although no significant difference was observed. The immunohistochemical results showed that both Ki-67 and NFIX were significantly increased in the hsa_circ_0005140 overexpression group, while significantly decreased in the knockdown group (Fig. 7 C). Table 1 Top 10 differentially expressed (upregulated and downregulated) circRNAs in Y79 cells compared with ARPE-19 cells. Chr_Start_End_Strand circBankID circbaseID splicedSeqLength Gene logFC foldChange PValue diffState chr2_116066815_116101488_+ hsa_circDPP10_008 hsa_circ_0117102 211 DPP10 11.24347595 2424.507421 1.79341E-66 up chr18_74083422_74092259_- hsa_circZNF516_004 hsa_circ_0005140 1555 ZNF516 9.220094303 596.382578 5.60346E-27 up chr2_55252222_55255356_- hsa_circRTN4_003 hsa_circ_0054598 2457 RTN4 9.190931613 584.4483054 6.32154E-28 up chr14_31346778_31349940_+ hsa_circCOCH_002 hsa_circ_0031431 547 COCH 9.104273862 550.3760426 6.70684E-26 up chr5_155281559_155297434_+ hsa_circSGCD_003 hsa_circ_0128413 SGCD 9.085816899 543.3797152 5.97392E-26 up chr2_15564439_15651474_- hsa_circNBAS_048 hsa_circ_0052762 1831 NBAS 8.970704653 501.7081808 2.17779E-25 up chr5_74130251_74137504_- hsa_circFAM169A_005 hsa_circ_0004405 493 FAM169A 8.936027503 489.7927142 1.20766E-24 up chr2_116094216_116101488_+ hsa_circDPP10_013 hsa_circ_0117108 DPP10 8.733644238 425.6855294 2.71786E-23 up chrX_147733520_147744289_+ hsa_circAFF2_003 hsa_circ_0091669 994 AFF2 8.713274108 419.7172967 8.94269E-23 up chr5_74109665_74137504_- hsa_circFAM169A_006 hsa_circ_0007158 673 FAM169A 8.677326944 409.3885505 8.39135E-23 up chr11_92085262_92088570_+ hsa_circFAT3_006 hsa_circ_0000348 3309 FAT3 -9.077299791 0.00185123 1.53262E-75 down chr4_81216713_81504337_+ FGF5,C4orf22 -9.188010531 0.001714483 1.875E-46 down chr7_158552177_158557544_- hsa_circESYT2_032 hsa_circ_0001776 495 ESYT2 -9.248356132 0.001644248 2.59965E-79 down chr6_16326625_16328701_- hsa_circATXN1_029 hsa_circ_0007132 2077 ATXN1 -9.362631192 0.001519033 3.68909E-48 down chr3_170906491_170912424_- hsa_circTNIK_010 hsa_circ_0002387 333 TNIK -9.525527554 0.001356846 2.45084E-52 down chr1_12638746_12639440_- hsa_circDHRS3_007 hsa_circ_0010023 359 DHRS3 -9.564885195 0.00132033 1.26132E-52 down chr5_49694941_49707217_- hsa_circEMB_003 hsa_circ_0001481 954 EMB -9.775013062 0.001141374 5.05181E-56 down chr4_81216713_81284038_+ hsa_circC4orf22_001 hsa_circ_0006205 FGF5,C4orf22 -10.05067341 0.000942857 4.6757E-63 down chr20_52773708_52788209_- hsa_circCYP24A1_012 hsa_circ_0060927 1106 CYP24A1 -10.92426063 0.0005146 3.27803E-87 down chr6_73005640_73043538_+ hsa_circRIMS1_046 hsa_circ_0132246 546 RIMS1 -11.24296149 0.000412602 1.29531E-96 down 4. Discussion RB demonstrates a bimodal heritability pattern, with hereditary and sporadic forms exhibiting distinct clinical manifestations. While hereditary RB manifests as either unilateral or bilateral disease, sporadic cases exclusively present unilaterally [ 45 ] . Although RB1 tumor suppressor gene inactivation constitutes the principal oncogenic driver, approximately 3–5% of RB cases develop through RB1-independent mechanisms, with rare instances demonstrating spontaneous tumor regression [ 46 ] . Our investigations identified hsa_circ_0005140 as a critical oncogenic circRNA in Y79 cells, where its overexpression significantly enhanced proliferative and invasive capacities through the miR-762/NFIX axis. Notably, this regulatory circuit also modulated proliferation in non-malignant ARPE-19 cells, implying evolutionary conservation in retinal homeostasis. Developmental tumors like RB may hijack such primordial pathways through circRNA overexpression, achieving constitutive pathway activation. In vivo validation confirmed these findings, with hsa_circ_0005140-overexpressing xenografts showing significantly increased tumor volume (p < 0.001). The dualistic nature of miR-762 emerges across pathological contexts: it exhibits tumor-suppressive effects in breast cancer by enhancing HDAC inhibitor-induced apoptosis [ 47 ] , yet promotes ovarian cancer progression via Wnt pathway activation [ 48 ] . In acute lung injury, miR-762-NFIX crosstalk regulates inflammatory responses [ 44 ] , mirroring our observation that hsa_circ_0005140 modulates IL-6/IL-8/TNF-α cytokine networks in RB. Mechanistically, hsa_circ_0005140 functions as a molecular sponge for miR-762 without inducing its degradation, evidenced by stable miR-762 levels despite circRNA manipulation. This sponge activity alters NFIX expression, which demonstrates context-dependent oncogenic roles - contrasting with its tumor-suppressive function in glioblastoma [ 49 ] . Functional assays confirmed that hsa_circ_0005140 knockdown induced apoptosis, reversible by miR-762 inhibition, paralleling NFIX mutation effects in Marshall-Smith syndrome [ 50 ] . Tumor xenograft experiments also indicated that the levels of miR-762 remained unchanged following the overexpression or knockdown of hsa_circ_0005140. These findings support the notion [ 51 ] that hsa_circ_0005140 acts as a sponge for miR-762 without leading to its degradation. These findings provide new insights not previously reported in the literature. As a pivotal developmental regulator, NFIX exhibits stage-specific and lineage-dependent functional characteristics. Particularly in retinal progenitor cells (RPCs), which serve as the cellular origin of RB, NFIX appears to play an indispensable role in sustaining proliferative capacity. The pathological foundation of RB stems from biallelic inactivation of the RB1 tumor suppressor gene, leading to comprehensive remodeling of cell cycle regulatory networks. Within this aberrant molecular framework, ‌NFIX may be co-opted by malignant cells through the hsa_circ_0005140/miR-762/NFIX axis‌, acquiring pro-proliferative properties that drive tumorigenesis. This mechanism parallels the oncogenic role of the ‌LINC00511/miR-625-5p/NFIX axis‌ in gastric cancer [52] , where LINC00511 overexpression suppresses miR-625-5p, thereby upregulating NFIX to promote proliferation. Such conserved regulatory patterns suggest NFIX's context-dependent hijacking as a common oncogenic driver across tumor types. Our data revealed that ‌NFIX upregulation was induced by hsa_circ_0005140 overexpression‌, exhibiting parallel increases with IL-6, IL-8, and TNF-α levels (p < 0.05). While these coordinated changes suggest potential functional linkages within the hsa_circ_0005140/miR-762/NFIX axis, ‌whether NFIX directly regulates these cytokines remains unresolved‌ and requires further mechanistic investigation. Although NFIX is a known transcriptional regulator, its binding to promoter regions of cytokine genes in this specific context needs experimental validation through future studies employing chromatin immunoprecipitation sequencing (ChIP-seq) and promoter-reporter assays. Limitations: This study primarily relied on one RB cell line and xenograft models; patient derived specimens were not assessed. The cytokine observations were correlative, and whether NFIX directly regulates cytokine transcription requires promoter reporter, ChIP-seq and luciferase reporter assays in subsequent studies. Future work will prioritize clinical validation and transcriptional mechanism mapping. 5. Conclusions This study reveals a circRNA-mediated regulatory axis (hsa_circ_0005140/miR-762/NFIX) that drives proliferation in retinoblastoma while maintaining retinal homeostasis. The conservation of this pathway in normal retinal cells suggests its developmental origin, which RB cells exploit via circRNA dysregulation. These findings highlight how non-coding RNAs can repurpose physiological networks for oncogenic progression, and with NFIX serving as a molecular switch whose oncogenic potential is determined by cellular context and RNA-mediated regulation. Abbreviations Age-related macular degeneration AMD Cell counting Kit-8 CCK-8 Complementary DNA cDNA Chromatin immunoprecipitation sequencing ChIP-seq Circular RNAs circRNAs Diabetic retinopathy DR Fetal bovine serum FBS microRNA miRNA nuclear factor I X gene NFIX Phosphate buffered saline PBS Propidium iodide PI Proliferative vitreoretinopathy PVR Retinoblastoma RB Retinal progenitor cells RPCs Real-time quantitative polymerase chain reaction RT-qPCR Western blotting WB Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The raw sequence data used for analysis are available in NCBI under the Sequence Read Archive (SRA), with the accession number SRP658189. Competing interests The authors declare that they have no competing interests. Funding The authors have no financial sponsorship from any company or institution. Authors' contributions Jihan Luo and Xiyuan Zhou contributed to the concept and study design. Jihan Luo completed all the experiments, made data interpretations, and drafted the manuscript. Xiyuan Zhou was involved in the critical revision of the manuscript, supervision of the manuscript, and final approval of the submission. ‌Hao Yao provided critical suggestions on manuscript revision, which significantly improved the clarity and scientific rigor of the paper. Acknowledgements Not applicable. References Li, Y. et al. Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis. Cell. Res. 25 (8), 981–984. 10.1038/cr.2015.82 (2015). Epub 2015 Jul 3. PMID: 26138677; PMCID: PMC4528056. Kristensen, L. S. et al. The biogenesis, biology and characterization of circular RNAs. Nat. Rev. Genet. 20 (11), 675–691. 10.1038/s41576-019-0158-7 (2019). Epub 2019 Aug 8. PMID: 31395983. Chen, L. L. 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09:43:43","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":289839,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/64a437df85947885510caf25.png"},{"id":100595925,"identity":"8b670c3d-8a5e-44bc-89a5-e60cc0fa2238","added_by":"auto","created_at":"2026-01-19 13:49:43","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":251630,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/3fa672aa1d32e0693161c7b7.png"},{"id":100595722,"identity":"81e3a4ff-8e06-4e18-8322-a6f71b31dd80","added_by":"auto","created_at":"2026-01-19 13:49:15","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":143628,"visible":true,"origin":"","legend":"","description":"","filename":"b479e1f943d54c87b942f311d83d9f7e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/e6d4520046483da199c0f675.xml"},{"id":100571342,"identity":"0f63ec06-9ef1-4aa6-98d0-d745fcc775ad","added_by":"auto","created_at":"2026-01-19 09:43:43","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":159964,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/feae2bebc5e1d92a74051f3f.html"},{"id":100571319,"identity":"b5a5f298-45e8-47d0-b450-adcce44eb8a3","added_by":"auto","created_at":"2026-01-19 09:43:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":385192,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential expression of circular RNA in Y79 and ARPE-19 cells: [A] Identification of circRNAs via high-throughput sequencing: The accompanying volcano plots display the distribution of gene expression levels. Here, the size of the dots represents the average expression value of the differentially expressed genes. The red dots represent genes whose expression was upregulated, the blue dots represent genes whose expression was downregulated, and the gray dots represent genes whose expression was not significantly changed. [B] miRNA-circRNA regulatory network: This panel depicts the regulatory network involving circ_0005140, with circular shapes indicating circRNAs and triangular shapes denoting miRNAs. In this schematic, red denotes upregulated components, and green signifies downregulated components. Validation of miR-762, NFIX, and selected circRNAs in Y79 and ARPE-19 cells via RT-qPCR: [C] and [D] These figures present the empirical validation and quantification of miR-762 and NFIX in Y79 and ARPE-19 cells. [E] The expression levels of the three most significantly upregulated circRNAs and the two most significantly downregulated circRNAs were validated and quantified. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001. Significance was determined by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/7d9aa21eef93e2950f1109f8.png"},{"id":100571323,"identity":"fd1de7d1-9b7a-44dd-81ad-f52d2e1f2ed2","added_by":"auto","created_at":"2026-01-19 09:43:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3647383,"visible":true,"origin":"","legend":"\u003cp\u003eCCK-8, EdU assays. Transwell assay for migration and invasion. [A] CCK-8 assay: The results demonstrated that hsa_circ_0005140 overexpression enhances the proliferation of ARPE-19 cells, whereas its knockdown suppresses their proliferation. Similarly, hsa_circ_0005140 overexpression increased the proliferation of Y79 cells, whereas hsa_circ_0005140 knockdown had an inhibitory effect. [B] [C] EdU assay: The data indicate that hsa_circ_0005140 overexpression significantly promotes the proliferation of ARPE-19 cells, whereas hsa_circ_0005140 knockdown results in reduced proliferation. In Y79 cells, the overexpression of hsa_circ_0005140 stimulates cell proliferation, whereas its knockdown decreases it. [D] Migration assay: Hsa_circ_0005140 overexpression significantly increased the migration capacity of ARPE-19 cells, whereas hsa_circ_0005140 knockdown diminished it. In Y79 cells, the overexpression of hsa_circ_0005140 enhances migration, whereas its knockdown inhibits this ability. [E] Invasion assay: Overexpression of hsa_circ_0005140 augments the invasive potential of ARPE-19 cells, whereas its knockdown inhibits it. In Y79 cells, hsa_circ_0005140 overexpression promotes invasion, whereas its knockdown leads to reduced invasion. Statistical significance: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, as assessed by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/c378a6af3fd8e3733cd32aee.png"},{"id":100595419,"identity":"61f29e54-1ce1-41a5-978a-fcb9d3ad16e6","added_by":"auto","created_at":"2026-01-19 13:48:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3396412,"visible":true,"origin":"","legend":"\u003cp\u003eCircRNA cellular function rescue assay. [A] Overexpression of miR-762 in CCK-8 assays negated the proliferation-promoting effect of hsa_circ_0005140 on ARPE-19 cells. Conversely, miR-762 knockdown inhibited the proliferation induced by hsa_circ_0005140 knockdown. Similar effects were observed in Y79 cells, where miR-762 overexpression reversed hsa_circ_0005140-induced proliferation, and knockdown of miR-762 restored the proliferation inhibited by hsa_circ_0005140 knockdown. [B] [C] EdU assay: miR-762 overexpression reversed the proliferation-promoting effects of hsa_circ_0005140 on ARPE-19 cells, while its knockdown reversed the inhibitory effects of hsa_circ_0005140 knockdown. This pattern was also evident in Y79 cells. [D] Transwell assay for migration: Overexpression of miR-762 mitigated the migration-enhancing effect of hsa_circ_0005140 on ARPE-19 cells, and miR-762 knockdown reversed the migration inhibition caused by hsa_circ_0005140 knockdown. Similar results were observed for Y79 cells. [E] Transwell assay for invasion: Overexpression of miR-762 reversed the invasion-promoting effects of hsa_circ_0005140 on ARPE-19 cells, whereas knockdown of miR-762 restored invasion inhibition caused by hsa_circ_0005140 knockdown. This effect was also replicated in Y79 cells. Statistical significance: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, as assessed by two-way ANOVA (A) or one-way ANOVA (B, C, D, E).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/0c04f9cdbbf550b9a07027c9.png"},{"id":100571325,"identity":"af199a05-0ab0-4215-81c0-9e91b0b8b77b","added_by":"auto","created_at":"2026-01-19 09:43:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":583572,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry analysis. [A] [B] Apoptosis assay: The results indicate that hsa_circ_0005140 overexpression inhibits apoptosis in ARPE-19 cells, whereas hsa_circ_0005140 knockdown facilitates apoptosis. In Y79 cells, hsa_circ_0005140 overexpression reduces apoptosis, whereas hsa_circ_0005140 knockdown increases it. [C] [D] Cell cycle assay: Overexpression of hsa_circ_0005140 decreases G0/G1 phase arrest in ARPE-19 cells, whereas hsa_circ_0005140 knockdown exacerbates G0/G1 phase arrest. In Y79 cells, the overexpression of hsa_circ_0005140 similarly reduces G0/G1 phase arrest, whereas its knockdown amplifies it. [E] [F] Apoptosis assay: Overexpression of miR-762 negated the inhibitory effect of hsa_circ_0005140 on apoptosis in ARPE-19 cells, whereas miR-762 knockdown reversed the proapoptotic effect of hsa_circ_0005140 knockdown. A similar trend was observed in Y79 cells. [G] [H] Cell cycle assay: miR-762 overexpression reversed the reduction in G0/G1 phase arrest in ARPE-19 cells caused by hsa_circ_0005140 overexpression, and knockdown of miR-762 reversed the increase in G0/G1 phase arrest caused by hsa_circ_0005140 knockdown. In Y79 cells, miR-762 overexpression mitigated G0/G1 phase arrest, whereas miR-762 knockdown reversed the inhibition of increased G0/G1 phase arrest. Statistical significance is noted as *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001, as determined by one-way ANOVA (E, F) or two-way ANOVA (A,B,C,D,G, H).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/7bfe33eb98b6056574f9ae2e.png"},{"id":100595926,"identity":"a92e8077-a9a2-4d2a-a8a7-c0e7f1484bb6","added_by":"auto","created_at":"2026-01-19 13:49:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":376454,"visible":true,"origin":"","legend":"\u003cp\u003eDual-luciferase reporter assay. [A] Compared with the NC group, the miR-762 mimic group transfected with the wild-type hsa_circ_0005140 reporter gene presented significantly reduced luciferase activity. No such reduction was observed in the mutant hsa_circ_0005140 reporter group. [B] For the wild-type NFIX 3'UTR reporter gene, miR-762 mimic overexpression significantly decreased luciferase activity compared with that in the NC group, whereas no significant change in the mutant NFIX 3'UTR reporter gene was detected. Statistical significance: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, as assessed by two-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/9e230777e4eab0934d7ae580.png"},{"id":100595915,"identity":"f1d9b94f-66b2-4eb4-9e57-53b6f11eedf8","added_by":"auto","created_at":"2026-01-19 13:49:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1424764,"visible":true,"origin":"","legend":"\u003cp\u003eXenograft assay. [A] Overexpression of hsa_circ_0005140 significantly enhanced tumor tissue growth, whereas miR-762 overexpression markedly attenuated this effect. The tumor weight in the hsa_circ_0005140 overexpression group was significantly greater than that in the control group, with miR-762 overexpression diminishing the growth-promoting effect of hsa_circ_0005140. [B] Knockdown of hsa_circ_0005140 significantly inhibited tumor growth, whereas miR-762 knockdown counteracted this inhibitory effect. The tumor weight in the hsa_circ_0005140 knockdown group was significantly lower than that in the control group, with miR-762 knockdown alleviating the growth suppression caused by hsa_circ_0005140 knockdown. [C] Overexpression of hsa_circ_0005140 upregulated NFIX expression in tumor tissue, which was significantly reversed by miR-762 overexpression. Conversely, hsa_circ_0005140 knockdown downregulated NFIX expression, whereas miR-762 knockdown reversed this effect. Furthermore, hsa_circ_0005140 overexpression did not significantly affect miR-762 levels, whereas miR-762 overexpression significantly increased miR-762 expression. In contrast, hsa_circ_0005140 knockdown had no significant effect on miR-762 levels, but miR-762 knockdown significantly altered its expression. Statistical significance is noted as *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001, as determined by one-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/a648bb202e3602dd2953c70c.png"},{"id":100571329,"identity":"6279544c-e09b-485f-8116-e7f872a6b09e","added_by":"auto","created_at":"2026-01-19 09:43:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":556681,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blotting. Full-length blots/gels are presented in Supplementary Figures.[A] Overexpression of hsa_circ_0005140 significantly elevated NFIX, IL-6, and IL-8 protein levels in Y79 cells, whereas overexpression of miR-762 reversed these increases. Conversely, knockdown of hsa_circ_0005140 led to marked decreases in NFIX, IL-6, and IL-8 expression, which were reversed by miR-762 knockdown. Neither the overexpression of hsa_circ_0005140 nor the expression of miR-762 significantly affected TNF-α protein levels in Y79 cells. However, hsa_circ_0005140 knockdown significantly reduced TNF-α expression, which was subsequently reversed by miR-762 knockdown. [B] Overexpression of hsa_circ_0005140 significantly elevated NFIX, IL-8 and TNF-α protein levels in ARPE-19 cells, with miR-762 overexpression reversing these effects. Conversely, knockdown of hsa_circ_0005140 markedly reduced the expression of NFIX, IL-8 and TNF-α, which was reversed by miR-762 knockdown. Overexpression of hsa_circ_0005140 also notably increased IL-6 protein levels in ARPE-19 cells, however, while knockdown of hsa_circ_0005140 significantly reduced IL-6 expression, miR-762 knockdown reversed this reduction, although no significant difference was observed. [C] Immunohistochemical assay: Ki-67 and NFIX were measured by immunohistochemical staining in the blank group, overexpression group, and knockdown group, the proportion of positive cells was quantified based on the immunostaining results. Statistical significance is noted as *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001, as determined by one-way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/9c30a5abd2762c274e4f0e03.png"},{"id":103507549,"identity":"c4b04ed7-8697-4b38-8f4f-3efb31ff02d2","added_by":"auto","created_at":"2026-02-26 13:41:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11204885,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/3053e5a4-9d5b-48c6-92ee-2297ac857f2a.pdf"},{"id":100595586,"identity":"6acc59f6-ec0a-4c2b-9a49-5d52bfa6a03d","added_by":"auto","created_at":"2026-01-19 13:48:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5299872,"visible":true,"origin":"","legend":"","description":"","filename":"GAPDHARPE19.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/bbfd97389b1fc34c008d6ddc.pdf"},{"id":100571320,"identity":"2911986b-f5e7-4230-8049-da3fa86d069b","added_by":"auto","created_at":"2026-01-19 09:43:43","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":58983,"visible":true,"origin":"","legend":"","description":"","filename":"file.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8392296/v1/d8652d92df988360f28dc72b.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oncogenic hijacking of a conserved hsa_circ_0005140/miR-762/NFIX axis drives retinoblastoma proliferation through context-dependent activation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCircular RNAs (circRNAs) represent a novel class of noncoding RNAs that regulate cellular activities. Characterized by their closed circular structure, circRNAs lack a 5' cap and 3' poly(A) tail structures, are predominantly found in the cytoplasm or exosomes, and are resistant to RNA exonucleases\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Their stability and resistance to degradation make them promising for various applications. Recent studies have highlighted the involvement of circRNAs in numerous biological processes, including cell growth, the stress response, and disease progression. They show considerable potential as diagnostic markers and therapeutic targets in cancers or other diseases, influencing processes such as cell viability, proliferation, apoptosis, autophagy, migration, and invasion.\u003c/p\u003e \u003cp\u003eCircRNAs are generally generated through postsplicing of precursor microRNA (miRNA) exons\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. On the basis of their original sequences, circRNAs are classified into three types: exon-intron, intron-intron, and exon-intron (EIcircRNA) types\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. The recognized biological functions of circRNAs include the following\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e: miRNA sponging, protein binding regulation, gene transcription regulation and encoding function.\u003c/p\u003e \u003cp\u003eThe most significant function of circRNAs is their role as miRNA sponges\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. CircRNAs with miRNA binding sites directly bind to miRNAs, inhibiting their function and regulating target gene expression through what is known as the circRNA ceRNA axis. This multifactorial interaction network suggests that targeting specific circRNAs could alter the levels of multiple disease-related RNAs simultaneously, making circRNAs valuable in the study of diagnosis and therapeutic targets for complex diseases\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn retinal cells, circRNAs are increasingly recognized as modulators of proliferation and inflammatory responses, yet their conserved versus cell-type-specific functions remain poorly characterized. In retinal physiology and pathology, circRNAs play pivotal roles through miRNA sponging and protein scaffolding mechanisms. They are implicated in several retinal disorders\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, including diabetic retinopathy (DR), glaucoma, proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD)\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, and ocular cancers such like retinoblastoma (RB)\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn DR, circRNAs like circ_0005015\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e and circHIPK3\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e regulate vascular dysfunction by sponging specific miRNAs (e.g., miR-519d-3p and miR-30a-3p) to modulate endothelial cell proliferation and angiogenesis. Similarly, cPWWP2A\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e and cZNF532\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e maintain vascular integrity by sequestering miR-579 and miR-29a-3p, respectively. In glaucoma, cZNF609 exhibits neuroprotective effects by derepressing miR-615 targets such as METRN, thereby mitigating retinal ganglion cell degeneration\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. PVR involves circ_0043144-driven ARPE-19 cell proliferation and cytokine secretion (CCL2, VEGF-A), contributing to epiretinal membrane formation\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. AMD pathogenesis is linked to circNR3C1 downregulation, which normally protects RPE cells via the miR-382-5p/PTEN/AKT/mTOR pathway against oxidative stress\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Three lncRNAs (ENSGALT00000098661, ENSGALT00000100816, and MSTRG.16980.1) and one circRNA (novel_circ_010168) in the ncRNA-mRNA regulatory network were identified as key molecules influencing circadian rhythm by regulating AOX1 in retinal metabolism\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Research on RB-related circRNAs has identified several oncogenic circRNAs, such as circ-FAM158A\u003csup\u003e[\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, circ-DHDDS\u003csup\u003e[\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, circ-E2F3\u003csup\u003e[\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, circ-TRHDE\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, circ-E2F5\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, circ-ODC1\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e, circ-RNF20\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e, and circ-0007534\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e, which promote disease progression and metastasis. Conversely, circ-TET1\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, circ-SHPRH\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e, circ-MKLN1\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e, and circ-CUL2\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e act as tumor suppressors in RB.\u003c/p\u003e \u003cp\u003eIn this study, we analyzed high-throughput sequencing data from Y79 retinoblastoma cells and ARPE-19 retinal pigment epithelial cells, and identified hsa_circ_0005140 as being markedly overexpressed in RB. We had designed a series of studies to explore its mechanism of action in RB. We confirmed the existence of the molecular regulatory axis of hsa_circ_0005140 /miR-762/NFIX. At the same time, cell function experiments revealed an unexpected phenomenon - the pro-proliferative function of this axis is not specific to RB tumor cells, but also exists in ARPE-19 cells. Through the analysis of these experimental data, we speculate that RB tumor cells may regulate a fundamental regulatory pathway crucial for retinal cell proliferation by abnormally upregulating hsa_circ_0005140, thereby driving their malignant proliferation.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003e2.1. Cell culture\u003c/h2\u003e\n \u003cp\u003eThe retinoblastoma cell line Y79 (ATCC HTB-18) RRID: CVCL_1895 and the normal retinal pigment epithelial cell line ARPE-19 (ATCC CRL-2302) RRID: CVCL_0145 were procured from Cellverse Co., Ltd. (Shanghai, China). Both cell lines were maintained in RPMI-1640 medium (Gibco, Los Angeles, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Opcel Biotechnology Co., Ltd., Inner Mongolia, China). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003e2.2. High-throughput sequencing\u003c/h2\u003e\n \u003cp\u003eHigh-throughput sequencing was performed on Y79 and ARPE-19 cells by GENESEED Biotechnology Co., Ltd. (Guangzhou, China). This sequencing aimed to generate circRNA expression profiles for retinoblastoma, identify the top 10 differentially expressed (upregulated and downregulated) circRNAs, and explore their downstream interaction networks. The target genes were also determined for further research. The raw sequence data used for analysis are available in NCBI under the Sequence Read Archive (SRA), with the accession number SRP658189.\u003c/p\u003e\n \u003ch2\u003e2.3. Real-time quantitative polymerase chain reaction (RT-qPCR)\u003c/h2\u003e\n \u003cp\u003eRNA was extracted from Y79 and ARPE-19 cells via TRIzol reagent (Biosharp, Beijing, China). Complementary DNA (cDNA) was synthesized via a reverse transcription kit(with dsDNase) from Biosharp. RT-qPCR was conducted using Universal SYBR qPCR Master Mix (Biosharp). The RNA expression levels were quantified via the 2 − ΔΔCt method, with GAPDH or U6 (for miR-762) serving as internal controls. Each sample was analyzed in triplicate. The following primers were used:\u003c/p\u003e\n \u003cp\u003ecirc_117102-F1: 5'-CGGTGGATCAATGGAACTGG-3', circ_117102-R1: 5'-ACTAAAATCACCAGCAGAGCA-3'\u003c/p\u003e\n \u003cp\u003ecirc_5140-F2: 5'-CACATGCGGGCACACTCA-3', circ_5140-R2: 5'-TTTCCGAGGCGTTATCTCCC-3'\u003c/p\u003e\n \u003cp\u003ecirc_54598-F2: 5'-CAAGCAGAGATAGAGAGCATAGT-3', circ_54598-R2: 5'-CTCAGATGCAGCAGGAAGA G-3'\u003c/p\u003e\n \u003cp\u003ecirc_60927-F1: 5'-GACATCCAGGCCACAGACAA-3', circ_60927-R1: 5'-CCAGTCTTCCCCTTCCCTGA-3'\u003c/p\u003e\n \u003cp\u003ecirc_132246-F1: 5'-CCCAGCTTAGTCAAACAGAACAA-3', circ_132246-R1: 5'-CTGTCTTGTTCGCTCCTCCA-3'\u003c/p\u003e\n \u003cp\u003eNFIX-F: 5'-GCCTTGACTCCTCCATCACC-3', NFIX-R: 5'-CAGGACTGAGACTGCTGTGG-3'\u003c/p\u003e\n \u003cp\u003emiR-762 RT: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTCGG, miR-762-F: 5'-GGGGCTGGGGCCGGGG-3', miR-762-R: 5'-AGTGCAGGGTCCGAGGTATT-3'\u003c/p\u003e\n \u003cp\u003emiR-762 mimics: 5'-GGGGCUGGGGCCGGGGCCGAGC-3'\u003c/p\u003e\n \u003cp\u003emimics NC: 5'-UCCUGUACUGAGCUGCCCCGAG-3'\u003c/p\u003e\n \u003cp\u003emiR-762 inhibitor: 5'-GCUCGGCCCCGGCCCCAGCCCC-3'\u003c/p\u003e\n \u003cp\u003einhibitor NC: 5'-UCUACUCUUUCUAGGAGGUUGUGA-3'\u003c/p\u003e\n \u003cp\u003eGAPDH-F: 5'-AGAAGGCTGGGGCTCATTTG-3', GAPDH-R: 5'-GCAGGAGGCATTGCTGATGAT-3'\u003c/p\u003e\n \u003cp\u003eU6-F: 5'-CTCGCTTCGGCAGCACA-3', U6-R: 5'-AACGCTTCACGAATTTGCGT-3'\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003e2.4. Construction of overexpression and interference plasmids\u003c/h2\u003e\n \u003cp\u003eThe full-length sequence (1555 bp) of hsa_circ_0005140 was amplified from cDNA via PCR and subsequently cloned and inserted into the pLC5 ciR vector via an In-Fusion one-step cloning kit (Yeasen, Shanghai, China). The sequences of primers used for amplification were as follows:\u003c/p\u003e\n \u003cp\u003eC5140-F: 5'-CATTATTTTTCTCTCTCTCGGGACAGCCGCCTGCT-3'\u003c/p\u003e\n \u003cp\u003eC5140-R: 5'-AGTATGGAGTTGTTAGCTAGGATCCAGTTGTTCTTACCTGAGTGTGCCCGCATGTGGG-3'\u003c/p\u003e\n \u003cp\u003eTo construct the interference plasmid, the shRNA sequences targeting 5140-Sh GGGCACACTCAGGGGGGGACA were as follows:\u003c/p\u003e\n \u003cp\u003eF: GATCCGGGCACACTCAGGGACATCAAGAGTCCCCCTGTGTGCCTTTTTG\u003c/p\u003e\n \u003cp\u003eR: AATTCAAAAAAGGGCACACTCAGGGGGACACTCTTGATGTCCCCCTGAGTGTGCCCG\u003c/p\u003e\n \u003cp\u003eThe BamHI and EcoRI restriction sites were included at the 5' and 3' ends, respectively. The annealed double-stranded primers were ligated into the pLVX-shRNA2 vector. Plasmid extraction was performed via an endotoxin-free plasmid extraction kit (Magen, Guangzhou, China), and transfections were conducted with Lipo8000™ Transfection Reagent (Beyotime, Shanghai, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003e2.5. Cell transfection and selection of stably transfected cell lines\u003c/h2\u003e\n \u003cp\u003e293T cells were used to produce viral particles containing the overexpression plasmid hsa_circ_0005140-pLC5-ciR and interference plasmid hsa_circ_0005140-pLVX-shRNA2, along with copackaging plasmids (psPAX.2, pMD2.G). The viral supernatant was collected, concentrated, and stored at -80°C. Target cells (5 × 10\u003csup\u003e5\u003c/sup\u003e cells/well) were seeded into 6-well plates and incubated at 37°C with 5% CO\u003csub\u003e2\u003c/sub\u003e. After 48 hours of viral infection, the infection efficiency was assessed via fluorescence microscopy. Stably transfected cell lines were selected with 2 µg/mL puromycin for 2–3 weeks.\u003c/p\u003e\n \u003ch2\u003e2.6. Cell counting Kit-8 (CCK-8) assay\u003c/h2\u003e\n \u003cp\u003eTo assess cell proliferation, a CCK-8 assay was performed via a CCK-8 kit (SparkJade, Shandong, China). Approximately 5×10³ cells per well were seeded into 96-well plates and allowed to adhere for 3 hours. At days 1, 2, 3, 4, and 5, 10 µL of CCK-8 solution was added to the wells. The cells were then incubated in a 5% CO₂ environment at 37°C for 30 minutes. The absorbance was measured at 450 nm via a microplate reader.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003e2.7. EdU assay\u003c/h2\u003e\n \u003cp\u003eCell proliferation was further evaluated via an EdU-594 Cell Proliferation Detection Kit (Labgic, Beijing, China). After EdU labeling for 6 hours, the cells were washed with phosphate buffered saline (PBS), fixed, and permeabilized. The cells subsequently underwent a click reaction, followed by Hoechst 33342 staining. The samples were then visualized under a fluorescence microscope, and images were captured.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003e2.8. Transwell analysis\u003c/h2\u003e\n \u003cp\u003eCell migration and invasion were analyzed via 24-well transwell chambers (Corning, NY, USA). After transfection, Y79 and ARPE-19 cells were placed in the upper chambers, while the lower chambers were filled with RPMI-1640 medium supplemented with 10% FBS. After 24 hours of incubation, the cells were fixed in methanol and stained with 0.1% crystal violet. The migrated cells were counted in five random fields under a microscope at 100× magnification. For invasion assays, transwell inserts were precoated with Matrigel (Corning).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e2.9. Flow cytometry\u003c/h2\u003e\n \u003cp\u003eForty-eight hours posttransfection, Y79 and ARPE-19 cells were collected, trypsinized, and washed with PBS. The cells were fixed in 80% ethanol for 2 hours, treated with RNase A, and stained with propidium iodide (PI). The cell cycle distribution was determined via FACScan flow cytometry. Apoptotic cells were measured via the Annexin V-PE/7-AAD apoptosis detection kit (KeyGen) according to the manufacturer's guidelines, and data were acquired via FACScan flow cytometry.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e2.10. Dual-luciferase reporter assay\u003c/h2\u003e\n \u003cp\u003eThe circ_0005140 sequence, containing either wild-type or mutant miR-762 binding sites, was cloned and inserted into the psiCHECK2 vector (Promega, Beijing, China) to generate circ_0005140 wt or circ_0005140 mut constructs. Similarly, NFIX 3'UTR sequences with wild-type or mutant binding sites were cloned and inserted into the same vector. Y79 cells were cotransfected with the constructed reporters and miR-762 or miR-NC. Luciferase activity was measured via the Dual-Lucy assay kit (Labic).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e2.11. circRNA cellular function rescue assay\u003c/h2\u003e\n \u003cp\u003eFunctional validation of the circ_0005140/miR-762/NFIX axis was performed through CCK-8 assays, EdU assays, transwell analysis, flow cytometry, and RT-qPCR, utilizing overexpression and knockdown of miR-762 (Tsingke Biotechnology Co., Ltd., Beijing, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e2.12. Western blotting assay (WB)\u003c/h2\u003e\n \u003cp\u003eY79 and ARPE-19 cells were lysed in RIPA buffer (Biosharp) to extract total protein, which was quantified via a BCA protein assay kit (SparkJade). Proteins (30 µg) were subjected to SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked overnight with the indicated antibodies against NFIX, TNF-α, IL-6, IL-8, or GAPDH and then incubated with HRP-conjugated secondary antibodies (Proteintech Group, Rosemont, IL, USA) for 2 hours. Detection was conducted via the use of enhanced chemiluminescence (ECL) reagent (FUDEBio, Hangzhou, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003e2.13. Xenograft assay\u003c/h2\u003e\n \u003cp\u003eThe xenograft assay, which was approved by the Animal Ethics Committee of Chongqing Medical University (IACUC-SAHCQMU-2024-00099), followed National Institutes of Health guidelines and the study was reported in accordance with ARRIVE guidelines. At the initiation of the experiment, the BALB/c nude mice were 5 weeks old with an average body weight of approximately 15 g. Y79 cells (1×10⁷) stably expressing various constructs (Y79-OE-NC, Y79-C5140-OE, Y79-C5140- OE + miR-762 mimics, Y79-SH-NC, Y79-C5140-SH, Y79-C5140-SH + miR-762 inhibitor) were injected subcutaneously into the right flanks of 5-week-old BALB/c nude mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd) (n = 5 per group). The tumor volume was recorded every two days. After three weeks, the mice were sacrificed, and the tumors were excised for weight measurement and analysis of the miR-762 and NFIX levels. Ki-67 and NFIX were also measured by immunohistochemical staining. The euthanasia method for BALB/c nude mice was performed as follows: the posterior part of the mouse's head was firmly grasped with the thumb and index finger of the left hand, followed by a downward pressure. Simultaneously, the mouse's tail was held with the right hand and pulled forcefully backward and upward to induce cervical dislocation, resulting in instantaneous death of the mouse.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003e2.14. Statistical analysis\u003c/h2\u003e\n \u003cp\u003eThe data are presented as the means ± standard deviations. Statistical significance was determined via Student’s t test for two-group comparisons or two-way ANOVA for multiple groups, implemented in GraphPad Prism 9 (GraphPad, San Diego, CA, USA). P \u0026lt; 0.05 was considered statistically significant. All experiments were conducted at least in triplicate.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Differential expression of circular RNA in Y79 cells and ARPE-19 cells.\u003c/h2\u003e \u003cp\u003eTo obtain the circRNA expression profile in retinoblastoma cells, we conducted high-throughput sequencing of Y79 and ARPE-19 cells, with ARPE-19 cells used as a control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We identified the top 10 circRNAs with significant differential expression (upregulated and downregulated) between Y79 and ARPE-19 cells (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, we focused on hsa_circ_0005140, the second most highly expressed circRNA, which has an appropriate molecular weight and binding site availability. The downstream interaction network involving miR-762 and NFIX (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), which was predicted via high-throughput sequencing, also has clinical relevance, as this signaling pathway has been validated in study of acute lung injury\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Consequently, we selected hsa_circ_0005140 as the target gene for further investigation into the role of the miR-762/NFIX signaling pathway in the occurrence and progression of retinoblastoma.\u003c/p\u003e \u003cp\u003eWe then utilized RT-qPCR to measure the levels of miR-762 and NFIX in both Y79 and ARPE-19 cells. The results indicated that miR-762 levels were significantly lower in Y79 cells than in ARPE-19 cells, whereas NFIX levels were significantly higher in Y79 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Additionally, we verified the expression levels of the top three overexpressed circRNAs (hsa_circ_0117102, hsa_circ_0005140, and hsa_circ_0054598) and the top two underexpressed circRNAs (hsa_circ_0132246 and hsa_circ_0060927) in Y79 and ARPE-19 cells via RT-qPCR, which confirmed the high-throughput sequencing results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e3.2 Hsa_circ_0005140 knockdown suppressed Y79 and ARPE-19 cell proliferation, migration and invasion and facilitated apoptosis. miR-762 suppression eliminated the effects of hsa_circ_0005140 knockdown on Y79 and ARPE-19 cells.\u003c/p\u003e \u003cp\u003eWe subsequently performed functional assays by transfecting 293T cells with either the overexpression plasmid hsa_circ_0005140-pLC5-ciR or the knockdown plasmid hsa_circ_0005140-pLVX-shRNA2, along with the packaging plasmids psPAX.2 and pMD2. G, to produce viral solutions. These viral solutions were transfected into Y79 and ARPE-19 cells, from which we obtained stable cell lines through puromycin selection.\u003c/p\u003e \u003cp\u003eNext, we validated the role of hsa_circ_0005140 and miR-762 in retinoblastoma through functional cell assays, including CCK-8, EdU, transwell migration, and invasion experiments, and the results demonstrated that the overexpression of hsa_circ_0005140 increased the proliferation, migration, and invasion of Y79 and ARPE-19 cells, whereas the knockdown of hsa_circ_0005140 inhibited these processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The overexpression of miR-762 counteracted the proliferation, migration, and invasion-promoting effects of hsa_circ_0005140 in Y79 and ARPE-19 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Conversely, knockdown of miR-762 reversed the inhibition of these processes caused by hsa_circ_0005140 knockdown.\u003c/p\u003e \u003cp\u003eFlow cytometry analysis of apoptosis and the cell cycle revealed that overexpression of hsa_circ_0005140 reduced apoptosis and decreased G0/G1 phase arrest in both cell lines, while knockdown promoted apoptosis and enhanced G0/G1 phase arrest (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD); miR-762 overexpression negated the inhibitory effects of hsa_circ_0005140 overexpression on apoptosis and G0/G1 phase arrest in both cell lines. Similarly, knockdown of hsa_circ_0005140 enhanced apoptosis and G0/G1 phase arrest, an effect that was reversed by miR-762 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). These findings suggest that hsa_circ_0005140 may negatively regulate miR-762 by directly targeting it.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3 hsa_circ_0005140 interacts with hsa-miR-762, and hsa-miR-762 interacts with the NFIX gene.\u003c/h2\u003e \u003cp\u003eA dual-luciferase reporter assay was used to validate the circ_0005140/miR-762/NFIX signaling pathway. Compared with NC transfection, transfection of the wild-type hsa_circ_0005140 reporter gene plasmid with miR-762 mimics significantly reduced luciferase activity. In contrast, no significant difference was observed in the mutant hsa_circ_0005140 plasmid group. These results suggest an interaction between hsa_circ_0005140 and miR-762 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Compared with the NC, the overexpression of miR-762 mimics significantly decreased the luciferase activity of the wild-type NFIX 3'UTR reporter gene plasmid. However, there was no significant reduction in luciferase activity in the mutant NFIX 3'UTR group. These findings indicate a potential interaction between NFIX and miR-762 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Hsa_circ_0005140 knockdown suppressed RB tumor growth in vivo.\u003c/h2\u003e \u003cp\u003eThe effect of hsa_circ_0005140 on RB progression in vivo was investigated via xenotransplantation assay. The excised tumors are displayed in Fig.\u0026nbsp;8A and 8B, and the tumor growth rates and weights were recorded. The overexpression of hsa_circ_0005140 significantly promoted tumor growth, with the tumor weight in the overexpression group exceeding that in the control group. Conversely, overexpression of miR-762 attenuated this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In contrast, knockdown of hsa_circ_0005140 notably inhibited tumor growth, resulting in significantly lower tumor weights than those in the control group. Additionally, knockdown of miR-762 mitigated the growth inhibition associated with hsa_circ_0005140 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Furthermore, hsa_circ_0005140 overexpression led to increased NFIX expression in tumor tissue, whereas miR-762 overexpression significantly reversed this effect. Conversely, hsa_circ_0005140 knockdown reduced NFIX expression, which was reversed by miR-762 knockdown. Notably, hsa_circ_0005140 overexpression and knockdown did not significantly affect miR-762 levels; however, miR-762 overexpression significantly increased its expression, whereas knockdown decreased it (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Western blotting analysis\u003c/h2\u003e \u003cp\u003eWB was employed to assess alterations in NFIX levels within the hsa_circ_0005140/miR-762/NFIX signaling pathway, along with variations in the levels of related inflammatory cytokines, including IL-6, IL-8, and TNF-α(Figure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The overexpression of hsa_circ_0005140 significantly increased the protein levels of NFIX, IL-6, and IL-8 in both Y79 and ARPE-19 cells, whereas the overexpression of miR-762 reversed these effects. Conversely, knockdown of hsa_circ_0005140 markedly reduced the expression of NFIX, IL-6, IL-8, and TNF-α in both cell lines, with miR-762 knockdown reversing these reductions. Notably, neither the overexpression of hsa_circ_0005140 nor the expression of miR-762 significantly affected TNF-α levels in Y79 cells; however, hsa_circ_0005140 knockdown significantly decreased TNF-α, which was reversed by miR-762 knockdown. While hsa_circ_0005140 knockdown significantly reduced IL-6 levels in ARPE-19 cells, miR-762 knockdown mitigated this decrease, although no significant difference was observed. The immunohistochemical results showed that both Ki-67 and NFIX were significantly increased in the hsa_circ_0005140 overexpression group, while significantly decreased in the knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTop 10 differentially expressed (upregulated and downregulated) circRNAs in Y79 cells compared with ARPE-19 cells.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChr_Start_End_Strand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ecircBankID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecircbaseID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003esplicedSeqLength\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003elogFC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003efoldChange\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePValue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ediffState\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr2_116066815_116101488_+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circDPP10_008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0117102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDPP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e11.24347595\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2424.507421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.79341E-66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr18_74083422_74092259_-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circZNF516_004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0005140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eZNF516\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9.220094303\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e596.382578\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.60346E-27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr2_55252222_55255356_-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circRTN4_003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0054598\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2457\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRTN4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9.190931613\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e584.4483054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.32154E-28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr14_31346778_31349940_+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circCOCH_002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0031431\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCOCH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9.104273862\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e550.3760426\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.70684E-26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr5_155281559_155297434_+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circSGCD_003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0128413\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSGCD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9.085816899\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e543.3797152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.97392E-26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr2_15564439_15651474_-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circNBAS_048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0052762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1831\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNBAS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.970704653\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e501.7081808\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.17779E-25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr5_74130251_74137504_-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circFAM169A_005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0004405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e493\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFAM169A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.936027503\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e489.7927142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.20766E-24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr2_116094216_116101488_+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circDPP10_013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0117108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDPP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.733644238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e425.6855294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.71786E-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echrX_147733520_147744289_+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circAFF2_003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0091669\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e994\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAFF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.713274108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e419.7172967\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.94269E-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr5_74109665_74137504_-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circFAM169A_006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0007158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e673\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFAM169A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.677326944\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e409.3885505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.39135E-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr11_92085262_92088570_+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circFAT3_006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0000348\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3309\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFAT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-9.077299791\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.00185123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.53262E-75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr4_81216713_81504337_+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFGF5,C4orf22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-9.188010531\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.001714483\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.875E-46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr7_158552177_158557544_-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circESYT2_032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0001776\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e495\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eESYT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-9.248356132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.001644248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.59965E-79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr6_16326625_16328701_-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circATXN1_029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0007132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eATXN1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-9.362631192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.001519033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.68909E-48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr3_170906491_170912424_-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circTNIK_010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0002387\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTNIK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-9.525527554\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.001356846\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.45084E-52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr1_12638746_12639440_-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circDHRS3_007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0010023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDHRS3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-9.564885195\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.00132033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.26132E-52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr5_49694941_49707217_-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circEMB_003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0001481\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e954\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEMB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-9.775013062\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.001141374\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.05181E-56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr4_81216713_81284038_+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circC4orf22_001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0006205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFGF5,C4orf22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-10.05067341\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.000942857\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.6757E-63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr20_52773708_52788209_-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circCYP24A1_012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0060927\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCYP24A1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-10.92426063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0005146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.27803E-87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echr6_73005640_73043538_+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehsa_circRIMS1_046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehsa_circ_0132246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRIMS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-11.24296149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.000412602\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.29531E-96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eRB demonstrates a bimodal heritability pattern, with hereditary and sporadic forms exhibiting distinct clinical manifestations. While hereditary RB manifests as either unilateral or bilateral disease, sporadic cases exclusively present unilaterally\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Although RB1 tumor suppressor gene inactivation constitutes the principal oncogenic driver, approximately 3\u0026ndash;5% of RB cases develop through RB1-independent mechanisms, with rare instances demonstrating spontaneous tumor regression\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur investigations identified hsa_circ_0005140 as a critical oncogenic circRNA in Y79 cells, where its overexpression significantly enhanced proliferative and invasive capacities through the miR-762/NFIX axis. Notably, this regulatory circuit also modulated proliferation in non-malignant ARPE-19 cells, implying evolutionary conservation in retinal homeostasis. Developmental tumors like RB may hijack such primordial pathways through circRNA overexpression, achieving constitutive pathway activation. In vivo validation confirmed these findings, with hsa_circ_0005140-overexpressing xenografts showing significantly increased tumor volume (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n\u003cp\u003eThe dualistic nature of miR-762 emerges across pathological contexts: it exhibits tumor-suppressive effects in breast cancer by enhancing HDAC inhibitor-induced apoptosis\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e, yet promotes ovarian cancer progression via Wnt pathway activation\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. In acute lung injury, miR-762-NFIX crosstalk regulates inflammatory responses\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e, mirroring our observation that hsa_circ_0005140 modulates IL-6/IL-8/TNF-\u0026alpha; cytokine networks in RB.\u003c/p\u003e\n\u003cp\u003eMechanistically, hsa_circ_0005140 functions as a molecular sponge for miR-762 without inducing its degradation, evidenced by stable miR-762 levels despite circRNA manipulation. This sponge activity alters NFIX expression, which demonstrates context-dependent oncogenic roles - contrasting with its tumor-suppressive function in glioblastoma\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. Functional assays confirmed that hsa_circ_0005140 knockdown induced apoptosis, reversible by miR-762 inhibition, paralleling NFIX mutation effects in Marshall-Smith syndrome\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTumor xenograft experiments also indicated that the levels of miR-762 remained unchanged following the overexpression or knockdown of hsa_circ_0005140. These findings support the notion\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e that hsa_circ_0005140 acts as a sponge for miR-762 without leading to its degradation. These findings provide new insights not previously reported in the literature.\u003c/p\u003e\n\u003cp\u003eAs a pivotal developmental regulator, NFIX exhibits stage-specific and lineage-dependent functional characteristics. Particularly in retinal progenitor cells (RPCs), which serve as the cellular origin of RB, NFIX appears to play an indispensable role in sustaining proliferative capacity.\u003c/p\u003e\n\u003cp\u003eThe pathological foundation of RB stems from biallelic inactivation of the RB1 tumor suppressor gene, leading to comprehensive remodeling of cell cycle regulatory networks. Within this aberrant molecular framework, \u0026zwnj;NFIX may be co-opted by malignant cells through the hsa_circ_0005140/miR-762/NFIX axis\u0026zwnj;, acquiring pro-proliferative properties that drive tumorigenesis. This mechanism parallels the oncogenic role of the \u0026zwnj;LINC00511/miR-625-5p/NFIX axis\u0026zwnj; in gastric cancer\u003csup\u003e[52]\u003c/sup\u003e, where LINC00511 overexpression suppresses miR-625-5p, thereby upregulating NFIX to promote proliferation. Such conserved regulatory patterns suggest NFIX\u0026apos;s context-dependent hijacking as a common oncogenic driver across tumor types.\u003c/p\u003e\n\u003cp\u003eOur data revealed that \u0026zwnj;NFIX upregulation was induced by hsa_circ_0005140 overexpression\u0026zwnj;, exhibiting parallel increases with IL-6, IL-8, and TNF-\u0026alpha; levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). While these coordinated changes suggest potential functional linkages within the hsa_circ_0005140/miR-762/NFIX axis, \u0026zwnj;whether NFIX directly regulates these cytokines remains unresolved\u0026zwnj; and requires further mechanistic investigation. Although NFIX is a known transcriptional regulator, its binding to promoter regions of cytokine genes in this specific context needs experimental validation through future studies employing chromatin immunoprecipitation sequencing (ChIP-seq) and promoter-reporter assays.\u003c/p\u003e\n\u003cp\u003eLimitations: This study primarily relied on one RB cell line and xenograft models; patient derived specimens were not assessed. The cytokine observations were correlative, and whether NFIX directly regulates cytokine transcription requires promoter reporter, ChIP-seq and luciferase reporter assays in subsequent studies. Future work will prioritize clinical validation and transcriptional mechanism mapping.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study reveals a circRNA-mediated regulatory axis (hsa_circ_0005140/miR-762/NFIX) that drives proliferation in retinoblastoma while maintaining retinal homeostasis. The conservation of this pathway in normal retinal cells suggests its developmental origin, which RB cells exploit via circRNA dysregulation. These findings highlight how non-coding RNAs can repurpose physiological networks for oncogenic progression, and with NFIX serving as a molecular switch whose oncogenic potential is determined by cellular context and RNA-mediated regulation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAge-related macular degeneration \u0026nbsp; AMD\u003c/p\u003e\n\u003cp\u003eCell counting Kit-8 \u0026nbsp;CCK-8\u003c/p\u003e\n\u003cp\u003eComplementary DNA \u0026nbsp; cDNA\u003c/p\u003e\n\u003cp\u003eChromatin immunoprecipitation sequencing \u0026nbsp;ChIP-seq\u003c/p\u003e\n\u003cp\u003eCircular RNAs \u0026nbsp;circRNAs\u003c/p\u003e\n\u003cp\u003eDiabetic retinopathy \u0026nbsp; DR\u003c/p\u003e\n\u003cp\u003eFetal bovine serum \u0026nbsp;FBS\u003c/p\u003e\n\u003cp\u003emicroRNA \u0026nbsp; miRNA\u003c/p\u003e\n\u003cp\u003enuclear factor I X gene \u0026nbsp;NFIX\u003c/p\u003e\n\u003cp\u003ePhosphate buffered saline \u0026nbsp;PBS\u003c/p\u003e\n\u003cp\u003ePropidium iodide \u0026nbsp;PI\u003c/p\u003e\n\u003cp\u003eProliferative vitreoretinopathy \u0026nbsp; PVR\u003c/p\u003e\n\u003cp\u003eRetinoblastoma \u0026nbsp;RB\u003c/p\u003e\n\u003cp\u003eRetinal progenitor cells \u0026nbsp;RPCs\u003c/p\u003e\n\u003cp\u003eReal-time quantitative polymerase chain reaction \u0026nbsp;RT-qPCR\u003c/p\u003e\n\u003cp\u003eWestern blotting \u0026nbsp;WB\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe raw sequence data used for analysis are available in NCBI under the Sequence Read Archive (SRA), with the accession number SRP658189.\u003c/p\u003e\n\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe authors have no financial sponsorship from any company or institution.\u003c/p\u003e\n\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eJihan Luo and Xiyuan Zhou contributed to the concept and study design. Jihan Luo completed all the experiments, made data interpretations, and drafted the manuscript. Xiyuan Zhou was involved in the critical revision of the manuscript, supervision of the manuscript, and final approval of the submission. \u0026zwnj;Hao Yao provided critical suggestions on manuscript revision, which significantly improved the clarity and scientific rigor of the paper.\u003c/p\u003e\n\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi, Y. et al. Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis. \u003cem\u003eCell. 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PMID: 31889903; PMCID: PMC6933746.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"circular RNA, sponge, Hsa_circ_0005140, miR-762, NFIX","lastPublishedDoi":"10.21203/rs.3.rs-8392296/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8392296/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Circular RNAs play pivotal roles in cellular regulation, yet their mechanisms in retinal cells remain incompletely understood. Through comparative analysis of Y79 retinoblastoma and ARPE-19 retinal pigment epithelial cells, hsa_circ_0005140 was identified as a functionally active circRNA engaging miR-762 and nuclear factor I X (NFIX).\u003c/p\u003e\n\u003cp\u003eMethods: Comprehensive functional assays included: RT-qPCR validation; Dual-luciferase reporter assays confirming molecular interactions; Phenotypic characterization (CCK-8/EdU/Transwell/Flow cytometry); Western blotting and xenograft models for in vivo validation‌.\u003c/p\u003e\n\u003cp\u003eResults: Differential expression, hsa_circ_0005140 showed 596-fold higher expression in Y79 than ARPE-19 (p\u0026lt;0.001), yet exhibited conserved regulatory functions in both cell types. Functional axis, acts as miR-762 sponge to upregulate NFIX (60% luciferase activity reduction, p\u0026lt;0.001). Proliferative effects, modulated cell cycle progression and apoptosis in both Y79 (p\u0026lt;0.01) and ARPE-19 (p\u0026lt;0.05). ‌Downstream effects, hsa_circ_0005140 overexpression led to elevated IL-6/IL-8 levels (2.1-3.5 fold changes) and context-dependent TNF-α responses. In Vivo Validation‌, Xenografts showed 2.3-fold tumor growth promotion by hsa_circ_0005140 (p\u0026lt;0.01), reversible by miR-762 overexpression.‌\u003c/p\u003e\n\u003cp\u003e‌Conclusions: This study establishes that ‌aberrant hsa_circ_0005140 overexpression activates a fundamental regulatory axis‌(circRNA/miR-762/NFIX) which exerts proliferative effects in both pathological and physiological contexts. The observed inflammatory marker dysregulation suggests broader functional consequences of circRNA dysregulation in retinal cells.\u003c/p\u003e","manuscriptTitle":"Oncogenic hijacking of a conserved hsa_circ_0005140/miR-762/NFIX axis drives retinoblastoma proliferation through context-dependent activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 09:43:38","doi":"10.21203/rs.3.rs-8392296/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"57f67b1f-6744-45d4-92c3-3ffedae5fe0b","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61288921,"name":"Biological sciences/Cancer"},{"id":61288922,"name":"Biological sciences/Cell biology"},{"id":61288923,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-04-12T06:55:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 09:43:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8392296","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8392296","identity":"rs-8392296","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}