circRNA Sequencing Research Articles

127aa encoded by circSpdyA promotes FA synthesis and NK cell repression in breast cancers.

Lipid metabolism reprogram plays key roles in breast cancer tumorigenesis and immune escape. The underlying mechanism and potential regulator were barely investigated. We thus established an in vivo tumorigenesis model, mice-bearing breast cancer cells were treated with an ordinary diet and high-fat diet, species were collected and subjected to circRNA sequence to scan the potential circRNAs regulating the lipid metabolism. CircSpdyA was one of the most upregulated circRNAs and had the potential to encode a 127-aa micro peptide (referred to as 127aa). 127 aa promotes tumorigenesis through promoting the fatty acid de novo synthesis by directly binding to FASN. Single-cell sequence indicated 127aa inhibited NK cell infiltration and function. This was achieved by inhibiting the transcription of NK cell activators epigenetically. Moreover, lipid-laden from 127aa positive cancer cells transferred to NK cells inhibited the cytotoxicity. Taken together, circSpdyA encoded 127aa promotes fatty acid de novo synthesis through directly binding with FASN and induced NK cell repression by inhibiting the transcription of NK cell activators.

CircUGP2 Suppresses Intrahepatic Cholangiocarcinoma Progression via p53 Signaling Through Interacting With PURB to Regulate ADGRB1 Transcription and Sponging miR-3191-5p.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer and its prognosis remains poor. Although growing numbers of studies have verified the involvement of circular RNAs (circRNAs) in various cancer types, their specific functions in ICC remain elusive. Herein, a circRNA, circUGP2 is identified by circRNA sequencing, which is downregulated in ICC tissues and correlated with patients' prognosis. Moreover, circUGP2 overexpression suppresses tumor progression in vitro and in vivo. Mechanistically, circUGP2 functions as a transcriptional co-activator of PURB over the expression of ADGRB1. It can also upregulate ADGRB1 expression by sponging miR-3191-5p. As a result, ADGRB1 prevents MDM2-mediated p53 polyubiquitination and thereby activates p53 signaling to inhibit ICC progression. Based on these findings, circUGP2 plasmid is encapsulated into a lipid nanoparticle (LNP) system, which has successfully targeted tumor site and shows superior anti-tumor effects. In summary, the present study has identified the role of circUGP2 as a tumor suppressor in ICC through regulating ADGRB1/p53 axis, and the application of LNP provides a promising translational strategy for ICC treatment.

Use of synthetic circular RNA spike-ins (SynCRS) for normalization of circular RNA sequencing data.

High-throughput RNA sequencing enables the quantification of transcript abundance and the identification of novel transcripts in biological samples. These include circular RNAs (circRNAs), a family of alternatively spliced RNA molecules that form a continuous loop. However, quantification and comparison of circRNAs between RNA sequencing libraries remain challenging due to confounding errors introduced during exonuclease digestion, library preparation and RNA sequencing itself. Here we describe a set of synthetic circRNA spike-ins-termed 'SynCRS'-that can be added directly to purified RNA samples before exonuclease digestion and library preparation. SynCRS, introduced either individually or in combinations of varying size and abundance, can be integrated into all next-generation sequencing workflows and, critically, facilitate the quantitative calibration of circRNA transcript abundance between samples, tissue types, species and laboratories. Our step-by-step protocol details the generation of SynCRS and guides users on the stoichiometry of SynCRS spike-in to RNA samples, followed by the bioinformatic steps required to facilitate quantitative comparisons of circRNAs between libraries. The laboratory steps to produce the SynCRS require an additional 3 d on top of the high throughput circRNA sequencing and bioinformatics. The protocol is suitable for users with basic experience in molecular biology and bioinformatics.

RNA-Seq Analysis Revealed circRNAs Associated with Resveratrol-Induced Apoptosis of Porcine Ovarian Granulosa Cells.

Circular RNAs (circRNAs) are a class of circular non-coding RNAs that play essential roles in the intricate and dynamic networks governing cell growth, development, and apoptosis. Resveratrol (RSV), a non-flavonoid polyphenol, is known to participate in follicular development and ovulation. In our previous research, we established a model using porcine ovarian granulosa cells (POGCs) treated with resveratrol, which confirmed its regulatory effects on long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) within these cells. However, the influence of resveratrol on circRNA expression has not been thoroughly investigated. To explore how resveratrol affects circRNA levels in POGCs, we designed an experiment with three groups: a control group (CON, n = 3, 0 μM RSV), a low-dose RSV group (LOW, n = 3, 50 μM RSV), and a high-dose RSV group (HIGH, n = 3, 100 μM RSV) for circRNA sequencing. We identified a total of 10,045 candidate circRNAs from POGCs treated with different concentrations of resveratrol (0, 50, and 100 μM). Differential expression analysis indicated that 96 circRNAs were significantly altered in the LOW vs. CON group, while 109 circRNAs showed significant changes in the HIGH vs. CON group. These circRNAs were notably enriched in biological processes associated with cell metabolism, apoptosis, and oxidative stress. Functional enrichment analysis of the host genes revealed their involvement in critical signaling pathways, including mTOR, AMPK, and apoptosis pathways. Additionally, we identified potential miRNA sponge candidates among the differentially expressed circRNAs, particularly novel_circ_0012954 and novel_circ_0004762, which exhibited strong connectivity within miRNA-target networks. Our findings provide valuable insights into the regulatory mechanisms of circRNAs in the context of resveratrol-induced apoptosis in POGCs, highlighting their potential as innovative therapeutic targets in reproductive biology.

CircRREB1 Mediates Metabolic Reprogramming and Stemness Maintenance to Facilitate Pancreatic Ductal Adenocarcinoma Progression.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal tumor with limited treatment options and poor patient survival. Circular RNAs (circRNAs) play crucial regulatory roles in the occurrence and development of various cancers, including PDAC. Here, using circRNA sequencing of diverse PDAC samples, we identified circRREB1 as an oncogenic circRNA that is significantly upregulated in PDAC and is correlated with an unfavorable patient prognosis. Functionally, loss of circRREB1 markedly inhibited glycolysis and stemness, while elevated circRREB1 elicited the opposite effects. Mechanistically, circRREB1 interacted with PGK1, disrupting the association between PTEN and PGK1 and increasing PGK1 phosphorylation to activate glycolytic flux. Moreover, circRREB1 promoted WNT7B transcription by directly interacting with YBX1 and facilitating its nuclear translocation, consequently activating the Wnt/β-catenin signaling pathway to maintain PDAC stemness. Overall, these results highlight circRREB1 as a key regulator of metabolic and stemness properties of PDAC.

Development and Validation of a Comprehensive Analysis of the Competing Endogenous circRNA/miRNA/mRNA Network for the Identification of Immune-Related Targets in Esophageal Squamous Cell Carcinoma.

Immunotherapy for esophageal squamous cell carcinoma (ESCC) exhibits notable variability in efficacy. Concurrently, recent research emphasizes circRNAs' impact on the ESCC tumor microenvironment. To further explore the relationship, we leveraged circRNA, microRNA, and mRNA sequence datasets to construct a comprehensive immune-related circRNA-microRNA-mRNA network, revealing competing endogenous RNA (ceRNA) roles in ESCC. The network comprises 16 circular RNAs, 13 microRNAs, and 1,560 mRNAs. Weighted gene co-expression analysis identified immune-related modules, notably cancer-associated fibroblast (CAF) and myeloid-derived suppressor cell modules, correlating significantly with immune and stemness scores. Among them, the CAF module plays a crucial role in extracellular matrix function and effectively discriminates ESCC patients. Four hub collagen family genes within CAF correlated robustly with CAF, macrophage infiltration, and T-cell exclusion. In-house sequencing and RT-qPCR validated their elevated expression. We also identified CAF module-targeting drugs as potential ESCC treatments. In summary, we established an immune-related circRNA-miRNA-mRNA network that not only illuminates ceRNA functionality but also highlights circRNAs' involvement in the CAF through collagen gene targeting. These findings hold promise to predict ESCC immune landscapes and therapy responses, ultimately aiding in more personalized and effective clinical decision-making.

Abstract We003: Identification of CircRNA-miRNA-mRNA Regulatory Network and Crucial Signaling Pathways Related to Cardiomyocyte Proliferation in Neonatal Mice

Introduction: Neonatal pig hearts exhibit remarkable regenerative capacity within two days post-birth. In this study, we propose that a comprehensive multiomics analysis encompassing circRNA, miRNA, and mRNA data from neonatal mouse hearts can unveil intricate circRNA-miRNA-mRNA relationships associated with cardiac regeneration. Our aim is to identify new novel circRNAs and miRNAs implicated in cardiomyocyte proliferation through leveraging multiomics analysis. Methods: Utilizing neonatal mouse hearts, we conducted circRNA, miRNA, and mRNA sequencing across four different postnatal days (P1, P3, P7, and P28; n=6, encompassing both males and females). We employed integrative bioinformatics analyses across distinct experimental designs (P1 vs P3, P1 vs P7, and P1 vs P28). Back splice junctions generated by CIRCexplorer2’s parse command was utilized to annotate sequenced circRNAs as known or novel. Validation of circRNA, miRNA, and mRNA expression levels was achieved through qRT-PCR, while protein expression levels were assessed via western blotting. Fluorescence in situ hybridization (FISH) technology was employed to determine circRNA localization. The functional significance of key genes involved in cardiomyocyte proliferation was assessed via siRNA-mediated knockdown of circRNAs and miRNAs, accompanied by cell cycle assessment using bioluminescence assay and immunostaining. Results: Intersection of differentially expressed target genes from circRNA, miRNA, and mRNA analyses at each experimental design facilitated the construction of circRNA-miRNA-mRNA regulatory networks, aiding in the development of cardiac regeneration-related regulatory networks and inference of novel relationships between circRNA, miRNA, and mRNA. Integrative bioinformatics analysis revealed significant alterations in circRNA-miRNA-mRNA expression profiles during postnatal cardiomyocyte maturation in mice. Pathway enrichment analysis identified several differentially expressed circRNAs enriched in cell division and proliferation-related pathways. qPCR and cell cycle studies identified 17 potential circRNAs associated with cardiomyocyte proliferation in mice, with knockdown of these circRNAs significantly impacting cardiomyocyte cell cycle. Conclusion: Our findings suggest a crucial role for circRNAs in the regulation of neonatal myocardial regeneration.

CircRHBDD1 promotes immune escape via IGF2BP2/PD-L1 signaling and acts as a nanotherapeutic target in gastric cancer

BackgroundCircular RNAs (circRNAs) have been implicated in the development and progression of gastric cancer (GC). However, it remains unclear whether dysregulated circRNA affects immune escape and the efficacy of immunotherapy in GC. Our aim is to investigate the molecular mechanism of circRNA affecting GC immunotherapy and identify effective molecular therapeutic targets.MethodsThe differential expression profile of circRNAs was established through circRNA sequencing, comparing three paired GC tissues with their adjacent non-cancerous gastric tissues. The expression level of circRHBDD1 in GC tissues was then assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The biological characteristics of circRHBDD1 were verified through a series of experiments, including agarose gel electrophoresis assays, RNase R treatment, and actinomycin D experiments. The prognostic value of circRHBDD1 in GC was evaluated by conducting both univariate and multivariate survival analyses. Furthermore, loss- and gain-of-function approaches were utilized to investigate the impact of circRHBDD1 on GC immune escape. RNA-sequencing, immunoprecipitation, flow cytometry, and methylated RNA immunoprecipitation (meRIP) analysis were performed to elucidate the underlying molecular mechanisms.ResultsWe discovered that circRHBDD1 exhibited remarkably high expression levels in GC tissues and cell lines. Notably, the high expression of circRHBDD1 was significantly correlated with poor overall survival and disease-free survival among GC patients. Both in vitro and in vivo experiments revealed that circRHBDD1 upregulated the expression of PD-L1 and impeded the infiltration of CD8+ T cells. Further, we found that circRHBDD1 binds to IGF2BP2, disrupting the interaction between E3 ligase TRIM25 and IGF2BP2, and ultimately inhibiting IGF2BP2 ubiquitination and degradation. Intriguingly, IGF2BP2 enhances PD-L1 mRNA stability through m6A modification. Additionally, we developed Poly (lactide-co-glycolic acid) (PLGA)-Polyethylene glycol (PEG)-based nanoparticles loaded with circRHBDD1 siRNA. In vivo experiments validated that the combination of PLGA-PEG(si-circRHBDD1) and anti-PD-1 offers a safe and efficacious nano-drug regimen for cancer immunotherapy.ConclusionOur results demonstrated that circRHBDD1 promoted GC immune escape by upregulating the expression of PD-L1 and reprogramming T cell-mediated immune response. Inhibition of circRHBDD1 expression could potentially enhance the response of GC patients to immunotherapy, thus improving treatment outcomes. Additionally, the development of a nanodrug delivery system provides a feasible approach for future clinical applications.

Identification and validation of key regulating circRNAs in Immune Thrombocytopenia by circRNAs sequencing

Dysfunction of T cells is a causative factor in Immune Thrombocytopenia (ITP), an autoimmune disorder. Circular RNAs (circRNAs), which have been associated with the pathophysiology of various immunological conditions, are of particular interest. Our study aimed to identify pivotal regulatory circRNAs within the peripheral T cells of ITP patients. We utilized circRNA sequencing to discern differences in circRNA expression between the ITP cohort and healthy controls. We identified 606 upregulated and 719 downregulated circRNAs. Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to scrutinize the parent genes of these differentially expressed circRNAs, revealing their significant involvement in metabolic processes and T cell receptor signaling pathways. Following a rigorous selection process that included DEG analysis, KEGG, and GO pathways analysis, along with an assessment of the potential roles of their parent genes, five top differentially expressed circRNAs were subjected to further validation via quantitative Polymerase Chain Reaction (RT-qPCR). Specifically, in the peripheral T cells of ITP patients, hsa_circ_0008866 (TAOK1), hsa_circ_0006856 (MAP3K5), and hsa_circ_0007444 (RHOBTB3) emerged as key regulatory circRNAs. The potential miRNA targets of these circRNAs were predicted employing miRanda, RNAhybrid, and TargetScan algorithms.

Identification and Functional Analysis of circRNAs during Goat Follicular Development.

Litter size is a crucial quantitative trait in animals, closely linked to follicular development. Circular RNA (circRNA), a type of single-stranded closed-loop endogenous RNA with stable expression, plays pivotal roles in various biological processes, yet its function in goat follicular development remains unclear. In this study, we collected large (follicle diameter > 3 mm) and small (1 mm < follicle diameter < 3 mm) follicles from black goats in the Chuanzhong region for circRNA sequencing, with the aim of elucidating the functional circRNAs that influence follicle development in goats. Differential analysis revealed that 17 circRNAs were upregulated in large follicles, and 28 circRNAs were upregulated in small follicles. Functional enrichment analysis revealed significant enrichment of pathways related to reproduction, including cellular response to follicle-stimulating hormone stimulus, the PI3K-Akt signaling pathway, the MAPK signaling pathway, and the Notch signaling pathway. Based on the ceRNA mechanism, 45 differentially expressed circRNAs were found to target and bind a total of 418 miRNAs, and an intercalation network including miR-324-3p (circRNA2497, circRNA5650), miR-202-5p (circRNA3333, circRNA5501), and miR-493-3p (circRNA4995, circRNA5508) was constructed. In addition, conservation analysis revealed that 2,239 circRNAs were conserved between goats and humans. Prediction of translation potential revealed that 154 circRNAs may potentially utilize both N6-methyladenosine (m6A) and internal ribosome entry site (IRES) translation mechanisms. Furthermore, the differential expression and circularization cleavage sites of five circRNAs were validated through RT-qPCR and DNA sequencing. Our study constructed a circRNA map in goat follicle development, offering a theoretical foundation for enhancing goat reproductive performance.

A novel protein encoded by circFOXP1 enhances ferroptosis and inhibits tumor recurrence in intrahepatic cholangiocarcinoma

CircRNAs participates in the development and occurrence of multiple tumor types. However, the specific effects and underlying mechanisms of circRNA in intrahepatic cholangiocarcinoma (ICC) progression and recurrence remain poorly understood. CircRNA sequencing was performed to screen circRNAs related to ICC recurrence after surgery using 53 ICC frozen tumor specimens. We found that compared with patients who experienced postsurgical recurrence, circFOXP1 had high expression in tumor tissues from patients with no postoperative recurrence. Functional experiments revealed that circFOXP1 inhibited ICC progression in vitro and in vivo. We then found that circFOXP1 inhibited ICC progression via encoding a novel protein, circFOXP1-231aa. Mechanistically, circFOXP1-231aa directly interacted with OTUD4, which regulates NCOA4 protein stability via deubiquitination modification, and thereby enhances ferroptosis of ICC cells. Examination of clinical ICC samples found positive correlations between circFOXP1 expression levels and levels of OTUD4 and NCOA4. These three factors are predictors of prognosis in patients with ICC. Collectively, we identified circFOXP1 encoded circFOXP1-231aa, which interacted with OTUD4 to suppress ubiquitination of NCOA4 and, thereby, promoted ferroptosis and inhibited ICC recurrence.

Induro-RT mediated circRNA-sequencing (IMCR-seq) enables comprehensive profiling of full-length and long circular RNAs from low input total RNA.

Circular RNA (circRNA) has recently gained attention for its emerging biological activities, relevance to disease, potential as biomarkers, and promising an alternative modality for RNA vaccines. Nevertheless, sequencing circRNAs has presented challenges. In this context, we introduce a novel circRNA sequencing method called Induro-RT mediated circRNA-sequencing (IMCR-seq), which relies on a group II intron reverse transcriptase with robust rolling circle reverse transcription activity. The IMCR-seq protocol eliminates the need for conventional circRNA enrichment methods such as rRNA depletion and RNaseR digestion yet achieved the highest circRNA enrichment and detected 6-1000 times more circRNAs for the benchmarked human samples compared to other methods. IMCR-seq is applicable to any organism, capable of detecting circRNAs of longer than 7000 nucleotides, and is effective on samples as small as 10 ng of total RNA. These enhancements render IMCR-seq suitable for clinical samples, including disease tissues and liquid biopsies. We demonstrated the clinical relevance of IMCR-seq by detecting cancer-specific circRNAs as potential biomarkers from IMCR-seq results on lung tumor tissues together with blood plasma samples from both a healthy individual and a lung cancer patient. In summary, IMCR-seq presents an efficient and versatile circRNA sequencing method with high potential for research and clinical applications.

Circular RNA hsa_circ_0005939 Regulates UHRF1BP1L Expression by Targeting miR-4693-3p to Promote Colorectal Cancer Progression.

Colorectal cancer (CRC) is the second most common and fatal cancer in China. circRNAs are different expressed between tumor and non-tumor tissues, and they are proved to be correlated with tumorigenesis and cancer progression. We aimed to explore the biological and molecular function of hsa_circ_0005939 in CRC. We collected and compared ten CRC tissues and four noncancerous tissues and performed circRNA sequencing. We investigated the hsa_circ_0005939 expression in fresh tissues from CRC and adjacent tissues by qPCR. Meanwhile, functional roles of hsa_circ_0005939 in CRC cells were explored by CCK-8, colony formation, wounding healing, cell apoptosis and western blot assays. RNA-FISH was used to confirm the cellular distribution of hsa_circ_0005939. Bioinformatic prediction and luciferase reporter assay were used to determine the mechanisms of hsa_circ_0005939. Our results indicated that hsa_circ_0005939 was up-regulated in CRC tissues and cells. Up-regulation of hsa_circ_0005939 was associated with the occurrence and the number of lymph node metastasis of CRC. Hsa_circ_0005939 down-regulation inhibited cell proliferation, increased cell apoptosis and caused G2 phase arrest of CRC cells. Mechanistically, luciferase assay revealed that hsa_circ_0005939 acts as a molecular sponge for miR-4693-3p and then enhanced Ubiquitin Like With PHD And Ring Finger Domains 1 binding protein 1 like (UHRF1BP1L) expression. Our findings indicated an oncogenic role of hsa_circ_0005939 in CRC, and it enhanced malignant phenotypes of CRC cells through miR-4693-3p/UHRF1BP1L axis. Our study may offer promising biomarkers and therapeutic targets for CRC.

BJLD-CMI: a predictive circRNA-miRNA interactions model combining multi-angle feature information.

Increasing research findings suggest that circular RNA (circRNA) exerts a crucial function in the pathogenesis of complex human diseases by binding to miRNA. Identifying their potential interactions is of paramount importance for the diagnosis and treatment of diseases. However, long cycles, small scales, and time-consuming processes characterize previous biological wet experiments. Consequently, the use of an efficient computational model to forecast the interactions between circRNA and miRNA is gradually becoming mainstream. In this study, we present a new prediction model named BJLD-CMI. The model extracts circRNA sequence features and miRNA sequence features by applying Jaccard and Bert's method and organically integrates them to obtain CMI attribute features, and then uses the graph embedding method Line to extract CMI behavioral features based on the known circRNA-miRNA correlation graph information. And then we predict the potential circRNA-miRNA interactions by fusing the multi-angle feature information such as attribute and behavior through Autoencoder in Autoencoder Networks. BJLD-CMI attained 94.95% and 90.69% of the area under the ROC curve on the CMI-9589 and CMI-9905 datasets. When compared with existing models, the results indicate that BJLD-CMI exhibits the best overall competence. During the case study experiment, we conducted a PubMed literature search to confirm that out of the top 10 predicted CMIs, seven pairs did indeed exist. These results suggest that BJLD-CMI is an effective method for predicting interactions between circRNAs and miRNAs. It provides a valuable candidate for biological wet experiments and can reduce the burden of researchers.

GEHGAN[formula omitted] CircRNA–disease association prediction via graph embedding and heterogeneous graph attention network

There is growing proof suggested that circRNAs play a crucial function in diverse important biological reactions related to human diseases. Within the area of biochemistry, a massive range of wet experiments have been carried out to find out the connections of circRNA–disease in recent years. Since wet experiments are expensive and laborious, nowadays, calculation-based solutions have increasingly attracted the attention of researchers. However, the performance of these methods is restricted due to the inability to balance the distribution among various types of nodes. To remedy the problem, we present a novel computational method called GEHGAN to forecast the new relationships in this research, leveraging graph embedding and heterogeneous graph attention networks. Firstly, we calculate circRNA sequences similarity, circRNA RBP similarity, disease semantic similarity and corresponding GIP kernel similarity to construct heterogeneous graph. Secondly, a graph embedding method using random walks with jump and stay strategies is applied to obtain the preliminary embeddings of circRNAs and diseases, greatly improving the performance of the model. Thirdly, a multi-head graph attention network is employed to further update the embeddings, followed by the employment of the MLP as a predictor. As a result, the five-fold cross-validation indicates that GEHGAN achieves an outstanding AUC score of 0.9829 and an AUPR value of 0.9815 on the CircR2Diseasev2.0 database, and case studies on osteosarcoma, gastric and colorectal neoplasms further confirm the model’s efficacy at identifying circRNA–disease correlations.

N6-methyladenosine-modified circSLCO1B3 promotes intrahepatic cholangiocarcinoma progression via regulating HOXC8 and PD-L1.

Refractoriness to surgical resection and chemotherapy makes intrahepatic cholangiocarcinoma (ICC) a fatal cancer of the digestive system with high mortality and poor prognosis. Important function invests circRNAs with tremendous potential in biomarkers and therapeutic targets. Nevertheless, it is still unknown how circRNAs contribute to the evolution of ICC. CircRNAs in paired ICC and adjacent tissues were screened by circRNAs sequencing. To explore the impact of circRNAs on ICC development, experiments involving gain and loss of function were conducted. Various experimental techniques, including quantitative real-time PCR (qPCR), western blotting, RNA immunoprecipitation (RIP), luciferase reporter assays, RNA pull-down, chromatin immunoprecipitation (ChIP), ubiquitination assays and so on were employed to identify the molecular regulatory role of circRNAs. Herein, we reported a new circRNA, which originates from exon 9 to exon 15 of the SLCO1B3 gene (named circSLCO1B3), orchestrated ICC progression by promoting tumor proliferation, metastasis and immune evasion. We found that the circSLCO1B3 gene was highly overexpressed in ICC tissues and related to lymphatic metastasis, tumor sizes, and tumor differentiation. Mechanically, circSLCO1B3 not only promoted ICC proliferation and metastasis via miR-502-5p/HOXC8/SMAD3 axis, but also eradicated anti-tumor immunity via suppressing ubiquitin-proteasome-dependent degradation of PD-L1 by E3 ubiquitin ligase SPOP. We further found that methyltransferase like 3 (METTL3) mediated the m6A methylation of circSLCO1B3 and stabilizes its expression. Our findings indicate that circSLCO1B3 is a potential prognostic marker and therapeutic target in ICC patients. Taken together, m6A-modified circSLCO1B3 was correlated with poor prognosis in ICC and promoted ICC progression not only by enhancing proliferation and metastasis via potentiating HOXC8 expression, but also by inducing immune evasion via antagonizing PD-L1 degradation. These results suggest that circSLCO1B3 is a potential prognostic marker and therapeutic target for ICC.

Magnetic Graphene Oxide Nanocomposites Boosts Craniomaxillofacial Bone Regeneration by Modulating circAars/miR-128-3p/SMAD5 Signaling Axis.

The ability of nanomaterials to induce osteogenic differentiation is limited, which seriously imped the repair of craniomaxillofacial bone defect. Magnetic graphene oxide (MGO) nanocomposites with the excellent physicochemical properties have great potential in bone tissue engineering. In this study, we aim to explore the craniomaxillofacial bone defect repairment effect of MGO nanocomposites and its underlying mechanism. The biocompatibility of MGO nanocomposites was verified by CCK8, live/dead staining and cytoskeleton staining. The function of MGO nanocomposites induced osteogenic differentiation of BMSCs was investigated by ALP activity detection, mineralized nodules staining, detection of osteogenic genes and proteins, and immune-histochemical staining. BMSCs with or without MGO osteogenic differentiation induction were collected and subjected to high-throughput circular ribonucleic acids (circRNAs) sequencing, and then crucial circRNA circAars was screened and identified. Bioinformatics analysis, Dual-luciferase reporter assay, RNA binding protein immunoprecipitation (RIP), fluorescence in situ hybridization (FISH) and osteogenic-related examinations were used to further explore the ability of circAars to participate in MGO nanocomposites regulation of osteogenic differentiation of BMSCs and its potential mechanism. Furthermore, critical-sized calvarial defects were constructed and were performed to verify the osteogenic differentiation induction effects and its potential mechanism induced by MGO nanocomposites. We verify the good biocompatibility and osteogenic differentiation improvement effects of BMSCs mediated by MGO nanocomposites. Furthermore, a new circRNA-circAars, we find and identify, is obviously upregulated in BMSCs mediated by MGO nanocomposites. Silencing circAars could significantly decrease the osteogenic ability of MGO nanocomposites. The underlying mechanism involved circAars sponging miR-128-3p to regulate the expression of SMAD5, which played an important role in the repair craniomaxillofacial bone defects mediated by MGO nanocomposites. We found that MGO nanocomposites regulated osteogenic differentiation of BMSCs via the circAars/miR-128-3p/SMAD5 pathway, which provided a feasible and effective strategy for the treatment of craniomaxillofacial bone defects.

Abstract 3348: Identification of circSPINT2 as a tumor suppressive circular RNA sensitizing lung cancer to 3rd generation EGFR-TKI treatment

Abstract Introduction: Lung cancer remains a high incidence and mortality globally. Adenocarcinoma is the most common type of lung cancer with around 50% of patients in Asia expressing mutated EGFR, which makes EGFR an effective therapeutic target to treat lung adenocarcinoma (LAC). Unfortunately, resistance to EGFR-TKI often occurs in patients through either dynamic EGFR mutation, driver oncogenic pathway shift, or transdifferentiating to evade EGFR-TKI treatment. circRNAs are a type of RNA molecule with a circular shape. Their importance in several biological processes and disease progression was intensively studied recently, and have emerged as promising diagnostic, prognostic, and monitoring biomarkers for diseases like cancer. CircRNAs’ unique circular structure and resistance to degradation make them robust candidates for non-invasive diagnostics of cancers. Aims: This study aimed to identify and investigate the involvement of a novel circular RNA (circSPINT2) in acquired resistance against the 3rd generation EGFR-TKI Osimertinib. Materials &amp; Methods: Comparative circRNA sequencing (circRNA-Seq), qRT-PCR, sanger sequencing, and RNase R treatment were applied to identify and verify the identity of Osimertinib-resistance-related circRNA. Biotinylated circRNA pull-down assay, miRNA-seq, and in-silico analysis were employed to delineate the downstream miRNA-mRNA regulatory axis. Osimertinib-resistant mouse xenograft model was established to explore the molecular mechanisms associated with both circSPINT2 and Osimertinib resistance. Results: We identified circSPINT2 as particularly downregulated in Osimertinib-resistant cell lines (OR) using circRNA-Seq. Overexpression of circSPINT2 in resistant cells increased cellular sensitivity to Osimertinib, whereas knockdown of circSPINT2 conferred Osimertinib resistance in parental cells. The circSPINT2-dependent regulation of Osimertinib sensitivity is mediated through modulating apoptosis. In-vitro analysis indicated high circSPINT2 increased tumor suppressor RBP1 expression by sequestering oncogenic hsa-miR-1296 further improving Osimertinib sensitivity. Molecular and histologic analysis of subcutaneous xenograft tumors corroborated our in-vitro results. Conclusion: Our study demonstrated circSPINT2 functions as a tumor suppressor, improving Osimertinib sensitivity via the hsa-miR-1296/RBP1 axis. circSPINT2 may serve as a prognostic and cancer biomarker to monitor Osimertinib resistance in LAC patients. Citation Format: Mong-Lien (Lotus) Wang, Shih-Hwa Shiou, Yi-Chen Chen, Ming-Long Tsai, Nalini Devi Verusingam, Alan Han-Kiat Ong. Identification of circSPINT2 as a tumor suppressive circular RNA sensitizing lung cancer to 3rd generation EGFR-TKI treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3348.

CircNUP54 promotes hepatocellular carcinoma progression via facilitating HuR cytoplasmic export and stabilizing BIRC3 mRNA

Circular RNAs (circRNAs) have been implicated in tumorigenesis and progression of various cancers. However, the underlying mechanisms of circRNAs in hepatocellular carcinoma (HCC) have not been fully elucidated. Herein, a new oncogenic circRNA, hsa_circ_0070039 (circNUP54), was identified to be significantly upregulated in HCC through circRNA sequencing. As verified in 68 HCC samples, circNUP54 overexpression was correlated with aggressive cancerous behaviors and poor outcomes. Moreover, the function experiments showed that knockdown of circNUP54 inhibited the malignant progression of HCC in vitro and in vivo, whereas overexpression of circNUP54 had the opposite role. Mechanistic investigations carried out by RNA pull-down, RNA immunoprecipitation, and immunofluorescence revealed that circNUP54 interacted with the RNA-binding protein Hu-antigen R (HuR) and promoted its cytoplasmic export. The cytoplasmic accumulation of HuR stabilized the downstream BIRC3 mRNA through its binding to the 3′ UTR region. Consequently, the encoded protein of BIRC3, cellular inhibitor of apoptosis 2 (cIAP2), proceeded to activate the NF-κB signal pathway and ultimately contributed to HCC progression. In addition, depletion of BIRC3 rescued the pro-tumorigenic effect of circNUP54 on HCC cells. Overall, this study demonstrated that circNUP54 facilitates HCC progression via regulating the HuR/BIRC3/NF-κB axis, which may serve as a promising therapeutic target for HCC treatment.

Predicting circRNA-RBP Binding Sites Using a Hybrid Deep Neural Network.

Circular RNAs (circRNAs) are non-coding RNAs generated by reverse splicing. They are involved in biological process and human diseases by interacting with specific RNA-binding proteins (RBPs). Due to traditional biological experiments being costly, computational methods have been proposed to predict the circRNA-RBP interaction. However, these methods have problems of single feature extraction. Therefore, we propose a novel model called circ-FHN, which utilizes only circRNA sequences to predict circRNA-RBP interactions. The circ-FHN approach involves feature coding and a hybrid deep learning model. Feature coding takes into account the physicochemical properties of circRNA sequences and employs four coding methods to extract sequence features. The hybrid deep structure comprises a convolutional neural network (CNN) and a bidirectional gated recurrent unit (BiGRU). The CNN learns high-level abstract features, while the BiGRU captures long-term dependencies in the sequence. To assess the effectiveness of circ-FHN, we compared it to other computational methods on 16 datasets and conducted ablation experiments. Additionally, we conducted motif analysis. The results demonstrate that circ-FHN exhibits exceptional performance and surpasses other methods. circ-FHN is freely available at https://github.com/zhaoqi106/circ-FHN .