ABSTRACT
BACKGROUND: Circular ribonucleic acids (circRNAs) are a type of covalently closed noncoding RNA with disease-relevant expressions, making them promising biomarkers for diagnosis and prognosis. Accurate quantification of circRNA in biological samples is a necessity for their clinical application. So far, methods developed for detecting circRNAs include northern blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), microarray analysis, and RNA sequencing. These methods generally suffer from disadvantages such as large sample consumption, cumbersome process, low selectivity, leading to inaccurate quantification of circRNA. It was thought that the above drawbacks could be eliminated by the construction of a microfluidic sensor. RESULTS: Herein, for the first time, a microfluidic sensor was constructed for circRNA analysis by using tetrahedral DNA nanostructure (TDN) as the skeleton for recognition probes and target-initiated hybridization chain reaction (HCR) as the signal amplification strategy. In the presence of circRNA, the recognition probe targets the circRNA-specific backsplice junction (BSJ). The captured circRNA then triggers the HCR by reacting with two hairpin species whose ends were labeled with 6-FAM, producing long DNA strands with abundant fluorescent labels. By using circ_0061276 as a model circRNA, this method has proven to be able to detect circRNA of attomolar concentration. It also eliminated the interference of linear RNA counterpart, showing high selectivity towards circRNA. The detection process can be implemented isothermally and does not require expensive complicated instruments. Moreover, this biosensor exhibited good performance in analyzing circRNA targets in total RNA extracted from cancer cells. SIGNIFICANCE: This represents the first microfluidic system for detection of circRNA. The biosensor showed merits such as ease of use, low-cost, small sample consumption, high sensitivity and specificity, and good reliability in complex biological matrix, providing a facile tool for circRNA analysis and related disease diagnosis in point-of care application scenes.
Subject(s)
DNA , Nanostructures , RNA, Circular , RNA, Circular/genetics , RNA, Circular/analysis , DNA/chemistry , Humans , Nanostructures/chemistry , Lab-On-A-Chip Devices , Nucleic Acid Hybridization , Biosensing Techniques/methods , Microfluidic Analytical Techniques/instrumentationABSTRACT
Circular RNAs (circRNAs) arise from precursor mRNA processing through back-splicing and have been increasingly recognized for their functions in various cancers including acute myeloid leukemia (AML). However, the prognostic implications of circRNA in AML remain unclear. We conducted a comprehensive genome-wide analysis of circRNAs using RNA-seq data in pediatric AML. We revealed a group of circRNAs associated with inferior outcomes, exerting effects on cancer-related pathways. Several of these circRNAs were transcribed directly from genes with established functions in AML, such as circRUNX1, circWHSC1, and circFLT3. Further investigations indicated the increased number of circRNAs and linear RNAs splicing were significantly correlated with inferior clinical outcomes, highlighting the pivotal role of splicing dysregulation. Subsequent analysis identified a group of upregulated RNA binding proteins in AMLs associated with high number of circRNAs, with TROVE2 being a prominent candidate, suggesting their involvement in circRNA associated prognosis. Through the integration of drug sensitivity data, we pinpointed 25 drugs that could target high-risk AMLs characterized by aberrant circRNA transcription. These findings underscore prognostic significance of circRNAs in pediatric AML and offer an alternative perspective for treating high-risk cases in this malignancy.
Subject(s)
Biomarkers, Tumor , Leukemia, Myeloid, Acute , RNA, Circular , Prognosis , Humans , Child , RNA, Circular/analysis , Biomarkers, Tumor/analysis , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Sequence Analysis, RNA , Datasets as Topic , Survival Analysis , K562 Cells , Progression-Free Survival , Treatment Outcome , Child, Preschool , Gene Expression Profiling , Gene Expression Regulation, LeukemicABSTRACT
BACKGROUND: Circular RNAs (circRNAs) have been confirmed to play a vital role in the occurrence and development of diseases. Exploring the relationship between circRNAs and diseases is of far-reaching significance for studying etiopathogenesis and treating diseases. To this end, based on the graph Markov neural network algorithm (GMNN) constructed in our previous work GMNN2CD, we further considered the multisource biological data that affects the association between circRNA and disease and developed an updated web server CircDA and based on the human hepatocellular carcinoma (HCC) tissue data to verify the prediction results of CircDA. RESULTS: CircDA is built on a Tumarkov-based deep learning framework. The algorithm regards biomolecules as nodes and the interactions between molecules as edges, reasonably abstracts multiomics data, and models them as a heterogeneous biomolecular association network, which can reflect the complex relationship between different biomolecules. Case studies using literature data from HCC, cervical, and gastric cancers demonstrate that the CircDA predictor can identify missing associations between known circRNAs and diseases, and using the quantitative real-time PCR (RT-qPCR) experiment of HCC in human tissue samples, it was found that five circRNAs were significantly differentially expressed, which proved that CircDA can predict diseases related to new circRNAs. CONCLUSIONS: This efficient computational prediction and case analysis with sufficient feedback allows us to identify circRNA-associated diseases and disease-associated circRNAs. Our work provides a method to predict circRNA-associated diseases and can provide guidance for the association of diseases with certain circRNAs. For ease of use, an online prediction server ( http://server.malab.cn/CircDA ) is provided, and the code is open-sourced ( https://github.com/nmt315320/CircDA.git ) for the convenience of algorithm improvement.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , RNA, Circular/genetics , RNA, Circular/analysis , Carcinoma, Hepatocellular/genetics , Follow-Up Studies , Liver Neoplasms/genetics , Neural Networks, Computer , Computer Simulation , Computational Biology/methodsABSTRACT
Labeled with pluripotent potential, the transplantation of bone marrow mesenchymal stem cells (BMSCs) is considered as a promising strategy for treating osteoporosis (OP). Melatonin (MEL) has been investigated to be an essential regulator involved in bone metabolism, as well as BMSCs differentiation. Circular RNAs (circRNAs) are a unique kind of non-coding RNA and play an important regulatory role in OP. However, whether circRNAs are implicated in the effects of MEL on BMSCs osteogenic differentiation remains largely indeterminate. Expression of circ_0005753 in human BMSCs with MEL treatment, clinical specimens diagnosed with OP, either with ovariectomy (OVX)-induced mice, was measured by RT-qPCR. Western blot was conducted to analyze protein levels of osteogenesis-related molecules (Opg, RUNX2, ALP, BMP4) and TXNIP. RNA immunoprecipitation (RIP) and RNA pull-down assays were performed to validate the binding relationship among circ_0005753, PTBP1, and TXNIP. Alkaline phosphatase (ALP) and alizarin red staining (ARS) were performed to evaluate osteogenic capacity of BMSCs. OP mouse model was established by ovariectomy, as evaluated pathologic changes via hematoxylin-eosin (HE), Masson, and Immunohistochemistry (IHC) staining. Expression of circ_0005753 was remarkably decreased during MEL-induced osteogenic differentiation of BMSCs. Interestingly, not only circ_0005753 knockdown significantly promoted osteogenic differentiation of BMSCs, but circ_0005753 overexpression also weakened osteogenic differentiation induced by MEL treatment. Mechanistically, circ_0005753 maintained the stabilization of TXNIP mRNA via recruiting PTBP1. Additionally, reinforced circ_0005753 abrogated MEL-mediated protective effects on OP pathogenesis in a mouse model. This work shows that MEL facilitates osteogenic differentiation of BMSCs via the circ_0005753/PTBP1/TXNIP axis, which may shed light on the development of a novel therapeutic strategy to prevent OP.
Subject(s)
Melatonin , Mesenchymal Stem Cells , MicroRNAs , Osteoporosis , Female , Mice , Humans , Animals , Osteogenesis , Melatonin/pharmacology , RNA, Circular/genetics , RNA, Circular/analysis , RNA, Circular/metabolism , Cells, Cultured , Osteoporosis/drug therapy , Osteoporosis/genetics , Cell Differentiation , Mesenchymal Stem Cells/metabolism , Disease Models, Animal , MicroRNAs/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/analysis , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Polypyrimidine Tract-Binding Protein/analysis , Polypyrimidine Tract-Binding Protein/metabolism , Polypyrimidine Tract-Binding Protein/pharmacology , Carrier Proteins/metabolismABSTRACT
Droplet digital polymerase chain reaction (ddPCR) is a new quantitative PCR method based on water-oil emulsion droplet technology. ddPCR enables highly sensitive and accurate quantification of nucleic acid molecules, especially when their copy numbers are low. In ddPCR, a sample is fractionated into ~20,000 droplets, and every nanoliter-sized droplet undergoes PCR amplification of the target molecule. The fluorescence signals of droplets are then recorded by an automated droplet reader. Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules that are ubiquitously expressed in animals and plants. CircRNAs are promising as biomarkers for cancer diagnosis and prognosis and as therapeutic targets or agents to inhibit oncogenic microRNAs or proteins (Kristensen LS, Jakobsen T, Hager H, Kjems J, Nat Rev Clin Oncol 19:188-206, 2022). In this chapter, the procedures for the quantitation of a circRNA in single pancreatic cancer cells using ddPCR are described.
Subject(s)
Biomarkers, Tumor , Polymerase Chain Reaction , RNA, Circular , Single-Cell Analysis , Single-Cell Analysis/instrumentation , Single-Cell Analysis/methods , RNA, Circular/analysis , RNA, Circular/genetics , Polymerase Chain Reaction/instrumentation , Polymerase Chain Reaction/methods , Pancreatic Neoplasms/diagnosis , Pancreatic Neoplasms/pathology , Cell Line, Tumor , Biomarkers, Tumor/analysis , HumansABSTRACT
Background: Circular RNAs (circRNAs) have been discovered in colorectal cancer (CRC), but there are few reports on the expression distribution and functional mining analysis of circRNAs. Methods: Differentially expressed circRNAs in CRC tissues and adjacent normal tissues were screened and identified by microarray and qRT-PCR. ROC curves of the six circRNAs were analyzed. A series of bioinformatics analyses on differentially expressed circRNAs were performed. Results: A total of 207 up-regulated and 357 down-regulated circRNAs in CRC were screened, and three top up-regulated and down-regulated circRNAs were chosen to be verified in 33 pairs of CRCs by qRT-PCR. 6 circRNAs showed high diagnostic values (AUC = 0.6860, AUC = 0.8127, AUC = 0.7502, AUC = 0.9945, AUC = 0.9642, AUC = 0.9486 for hsa_circRNA_100833, hsa_circRNA_103828, hsa_circRNA_103831 and hsa_circRNA_103752, hsa_circRNA_071106, hsa_circRNA_102293). A circRNA-miRNA-mRNA regulatory network (cirReNET) including six candidate circRNAs, 19 miRNAs and 210 mRNA was constructed, and the functions of the cirReNET were predicted and displayed via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on these mRNAs and protein-protein interaction (PPI) network of the hub genes acquired by string and CytoHubba. Conclusion: A cirReNET containing potential diagnostic and predictive indicators of CRCs and several critical circRNA-miRNA-mRNA regulatory axes (cirReAXEs) in CRC were mined, and may provide a novel route to study the mechanism and clinical targets of CRC.
Subject(s)
Biomarkers, Tumor , Colorectal Neoplasms , MicroRNAs , RNA, Circular , Humans , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/genetics , MicroRNAs/analysis , MicroRNAs/genetics , Protein Interaction Maps , RNA, Circular/analysis , RNA, Circular/genetics , RNA, Messenger/analysis , RNA, Messenger/genetics , Biomarkers, Tumor/analysis , Biomarkers, Tumor/geneticsABSTRACT
BACKGROUND: Sepsis is associated with high platelet turnover and elevated levels of immature platelets. Changes in the platelet transcriptome and the specific impact of immature platelets on the platelet transcriptome remain unclear. Thus, this study sought to address whether and how elevated levels of immature platelets affect the platelet transcriptome in patients with sepsis. METHODS: Blood samples were obtained from patients with sepsis requiring vasopressor therapy (n = 8) and from a control group of patients with stable coronary artery disease and otherwise similar demographic characteristics (n = 8). Immature platelet fraction (IPF) was determined on a Sysmex XE 2100 analyser and platelet function was tested by impedance aggregometry. RNA from leukocyte-depleted platelets was used for transcriptome analysis by Next Generation Sequencing integrating the use of unique molecular identifiers. RESULTS: IPF (median [interquartile range]) was significantly elevated in sepsis patients (6.4 [5.3-8.7] % vs. 3.6 [2.6-4.6] %, p = 0.005). Platelet function testing revealed no differences in adenosine diphosphate- or thrombin receptor activating peptide-induced platelet aggregation between control and sepsis patients. Putative circular RNA transcripts were decreased in platelets from septic patients. Leukocyte contamination defined by CD45 abundance levels in RNA-sequencing was absent in both groups. Principal component analysis of transcripts showed only partial overlap of clustering with IPF levels. RNA sequencing showed up-regulation of 524 and down-regulation of 118 genes in platelets from sepsis patients compared to controls. Upregulated genes were mostly related to catabolic processes and protein translation. Comparison to published platelet transcriptomes showed a large overlap of changes observed in sepsis and COVID-19 but not with reticulated platelets from healthy donors. CONCLUSIONS: Patients with sepsis appear to have a less degraded platelet transcriptome as indicated by increased levels of immature platelets and decreased levels of putative circular RNA transcripts. The present data suggests that increased protein translation is a characteristic mechanism of systemic inflammation.
Subject(s)
Blood Platelets/metabolism , Sepsis/genetics , Transcriptome/genetics , Aged , Base Sequence/genetics , Blood Platelets/pathology , Cell Fractionation/methods , Gene Expression/genetics , Gene Expression Profiling/methods , Humans , Male , Platelet Activation/genetics , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Platelet Count , Platelet Function Tests , RNA, Circular/analysis , RNA, Circular/genetics , Sepsis/blood , Sequence Analysis, RNA/methodsABSTRACT
BACKGROUND: Nowadays, non-invasive and rapid detection of cancers through molecular biomarkers has received much attention. Therefore, this study investigated the non-invasive and rapid diagnosis of colorectal cancer through one of the newest biomarkers (circular RNA). METHODS: For this purpose, we collected tumoral, adjacent normal tissue, and plasma samples from 100 colorectal cancer (CRC) patients, 25 postoperative CRC patients, 28 colitis patients, and 108 healthy donors. First Illumina high-throughput (Hi Seq 2000) sequencing was performed to identify known and novel differentially expressed circRNAs in the cancerous and adjacent normal tissues (n = 3). We used quantitative real-time fluorescent polymerase chain reaction (qRT-PCR) to detect the expression level of hsa_circ_0006282 among the different samples. Moreover, inter- and intra-assays were performed to evaluate the potential of hsa_circ_0006282 as being a biomarker. The receiver operating characteristic curve (ROC) was drawn to appraise its diagnostic efficacy, and the sensitivity of this circ RNA was evaluated. RESULTS: Based on RNA-sequencing results circ_0006282, cirs7, circ-0001313, circ_0055625, circ_000984, circ_0055625, circ_0001178, circ_0071589, circ-001569 were upregulated, and circ-ITGA7, circ-CDYL, circITCH, circ_0026344, circ_0000038, circ_0002220, circ_0067480, circIGHV3-20-1, circ_104916, circ_0009361 were downregulated circRNA. The hsa_circ_0006282 was the highest upregulated differentially expressed circRNA. Expression evaluation of this circRNA on different samples showed upregulation in CRC tissues (p < 0.0001) and plasma samples of CRC patients in comparison to healthy controls (p < 0.0001), while the area under the curve (AUC) was 0.831 (95% CI: 0.779-0.883). Expression of hsa_circ_0006282 in CRC patients decreased to normal after surgery (p < 0.0001). Our results showed high specificity and sensitivity of CRC detection when hsa_circ_0006282, carcinoembryonic antigen (CEA), and carbohydrate antigen199 (CA199) are combined. CONCLUSION: Plasma hsa_circ_0006282 can be used as a novel diagnostic and dynamic monitoring biomarker in CRC patients.
Subject(s)
Colorectal Neoplasms , RNA, Circular/blood , Biomarkers, Tumor/blood , Colon/pathology , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/epidemiology , Colorectal Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , Male , Middle Aged , RNA, Circular/analysis , Real-Time Polymerase Chain ReactionABSTRACT
The type of myofiber is related to the quality of meat. The slow oxidized myofiber helps to increase the tenderness and juiciness of muscle. Numerous studies have shown that circRNA plays a key role in skeletal muscle development. However, the role of circRNA in porcine skeletal myofiber types is unclear. In this study, we performed high-throughput RNA sequencing to study the differential expression of circRNA in the longissimus dorsi and the soleus muscle. A total of 40,757 circRNAs were identified, of which 181 were significantly different. Interestingly, some circRNAs were involved in metabolism pathways, AMPK, FoxO, and PI3K-Akt signaling pathways. Besides, we focused on a novel circRNA-circMYLK4. By injecting circMYLK4-AAV into piglets, we found that circMYLK4 significantly increased the mRNA and protein levels of the slow muscle marker genes. In summary, our study laid an essential foundation for further research of circRNA in myofiber type conversion and higher meat quality.
Subject(s)
Muscle Development/genetics , Muscle, Skeletal/growth & development , RNA, Circular/physiology , Swine , Animals , Animals, Genetically Modified , Cell Differentiation/genetics , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Male , Muscle Fibers, Fast-Twitch/physiology , Muscle Fibers, Slow-Twitch/physiology , Muscle, Skeletal/physiology , RNA, Circular/analysis , RNA, Circular/genetics , Swine/genetics , Swine/growth & developmentABSTRACT
BACKGROUND: Androgenetic alopecia (AGA) is an androgen-dependent polygenic hereditary disease. METHODS: Diseased hair follicles from 5 AGA patients and normal hair follicles from 5 healthy individuals were selected as specimens to carry out whole transcriptome sequencing. Multiple high-expression circular RNAs (circRNAs) were screened from the diseased group. We further verified the presence of the circRNAs in the clinical specimens by real-time fluorescence quantitative PCR (qRT-PCR). RESULTS: In total, 100 circRNAs were significantly upregulated, and 184 circRNAs were significantly downregulated. The top 10 upregulated circRNAs were hsa_circ_0101041, hsa_circ_0001578, hsa_circ_0135062, hsa_circ_0002980, hsa_circ_0005062, hsa_circ_0072688, hsa_circ_0133954, hsa_circ_0001079, hsa_circ_0005974, hsa_circ_0000489. The top 10 downregulated circRNAs were hsa_circ_0001278, hsa_circ_0031482, hsa_circ_0008285, hsa_circ_0003784, hsa_circ_0077096, hsa_circ_0001148, hsa_circ_0006886, hsa_circ_0000638, hsa_circ_0084521, and hsa_circ_0101074. Among top 10 upregulated circRNAs, hsa_circ_0001079 showed significantly high expression via large-sample verification and clinical application potential. Based on a database comparison and base pairing analysis, we found that has-miR-136-5p bound to hsa_circ_0001079 and that hsa-miR-136-5p had potential binding sites with Wnt5A. CONCLUSION: In summary, through high-throughput sequencing and bioinformatic analysis, a potential diagnostic marker for alopecia and a key circRNA that might adsorb microRNA (miRNA) through a sponging mechanism, thus mediating alopecia, were discovered in this study.
Subject(s)
Alopecia , MicroRNAs/genetics , RNA, Circular/genetics , Adult , Alopecia/genetics , Alopecia/metabolism , Alopecia/pathology , Biomarkers/metabolism , Computational Biology , Hair Follicle/chemistry , Hair Follicle/metabolism , High-Throughput Nucleotide Sequencing , Humans , Male , MicroRNAs/metabolism , RNA, Circular/analysis , RNA, Circular/metabolism , Transcriptome/genetics , Wnt-5a Protein/genetics , Wnt-5a Protein/metabolismABSTRACT
Circular RNAs (circRNAs), a novel type of noncoding RNAs, have been demonstrated to behave as microRNA (miRNA) sponges to exert their effects during pathological processes of diseases. However, the roles of circRNAs have not been explored in necrotizing enterocolitis (NEC). This study sought to identify differentially expressed circRNAs and predict their potential biological functions in NEC. circRNA expression profiles in terminal ileum from newborn rats with NEC and normal controls were explored using next-generation sequencing. In the NEC group, 53 circRNAs were significantly differentially expressed, including 9 upregulated and 44 downregulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted, and circRNA-miRNA interaction networks were generated to predict the potential roles of circRNAs in NEC progression. Further investigation revealed that most circRNAs include miRNA binding sites and that some are implicated in NEC development. In conclusion, this study's findings demonstrate that differentially expressed circRNAs are involved in NEC development via their interactions with miRNAs, making them prospective targets for NEC diagnosis and treatment.
Subject(s)
Enterocolitis, Necrotizing/genetics , RNA, Circular/genetics , Animals , Animals, Newborn , Computational Biology/methods , Enterocolitis, Necrotizing/metabolism , Gene Expression/genetics , Gene Expression Profiling/methods , Gene Expression Regulation/genetics , Gene Ontology , Gene Regulatory Networks/genetics , High-Throughput Nucleotide Sequencing/methods , Ileum/metabolism , MicroRNAs/genetics , Prospective Studies , RNA, Circular/analysis , Rats , Rats, Sprague-Dawley , Transcriptome/geneticsABSTRACT
Pneumonia caused by Mycoplasma pneumoniae (M. pneumoniae) is a major cause of communityacquired pneumonia in children. In some cases, M. pneumoniae pneumonia (MPP) can develop into refractory MPP (RMPP), which shows no clinical or radiological response to macrolides, and can progress to severe and complicated pneumonia. However, the pathogenesis of RMPP remains poorly understood. The present study aimed to identify target genes that could be used as biomarkers for the clinical diagnosis of earlystage RMPP through highthroughput sequencing technology. The differences in long noncoding (lnc)RNAs, mRNAs and circular (circ)RNAs were examined between wholeblood samples from two patients with nonrefractory MPP (NRMPP), two patients with RMPP and three healthy children using ribosomal (r)RNAdepleted RNAsequencing techniques and an integrated mRNA/circRNA analysis. A total of 17 lncRNAs (four upregulated and 13 downregulated), 18 mRNAs (six upregulated and 12 downregulated) and 24 circRNAs (12 upregulated and 12 downregulated) were the most significantly differentially expressed (P<0.05) between the NRMPP and RMPP groups. Upon functional analysis, the significantly differentially expressed genes encoded by the targeting mRNAs (prostaglandinendoperoxide synthase 2, IL8 and foslike antigen 1) were screened and identified to be enriched in the 'IL17 signaling pathway'. Furthermore, the key circRNAs in the NRMPP and RMPP comparative groups were primarily enriched in 'herpes simplex virus 1 infection', 'viral carcinogenesis' and 'RNA transport'. In the present study, a comprehensive analysis of the differences between the NRMPP and RMPP cases was performed based on rRNAdepleted RNAsequencing techniques, and the selected genes and circRNAs may be closely associated with the complex pathogenesis of RMPP.
Subject(s)
Anti-Bacterial Agents/pharmacology , Community-Acquired Infections/drug therapy , Drug Resistance, Bacterial/genetics , Mycoplasma pneumoniae/pathogenicity , Pneumonia, Mycoplasma/drug therapy , Anti-Bacterial Agents/therapeutic use , Biomarkers/analysis , Child , Child, Preschool , Community-Acquired Infections/genetics , Community-Acquired Infections/immunology , Community-Acquired Infections/microbiology , Female , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Infant , Macrolides/pharmacology , Macrolides/therapeutic use , Male , Pneumonia, Mycoplasma/genetics , Pneumonia, Mycoplasma/immunology , Pneumonia, Mycoplasma/microbiology , RNA, Circular/analysis , RNA, Long Noncoding/analysis , RNA, Messenger/analysis , RNA, Ribosomal , Sequence Analysis, RNA/methodsABSTRACT
PURPOSE: Circular RNA as a competitive endogenous RNA (ceRNA) plays a significant role in the pathogenesis and progression of breast cancer. In this study, a circular RNA-related ceRNA regulatory network was constructed, which provides new biomarkers and therapeutic targets for the treatment of breast cancer. Materials and methods. The expression profile datasets (GSE101123, GSE143564, GSE50428) of circRNAs, miRNAs, and mRNAs were downloaded from the GEO database, and then differentially expressed RNAs (DEcircRNAs, DEmiRNAs, DEmRNAs) were obtained through the CSCD, TargetScan, miRDB, and miRTarBase databases. CircRNA-miRNA pairs and miRNA-mRNA pairs were constructed. Finally, a ceRNA regulatory network was established. Downstream analysis of the ceRNA network included GO, KEGG analysis, survival analysis, sub-network construction, the BCIP, and qRT-PCR verification. RESULTS: In total, 144 differentially expressed (DE) DEcircRNA, 221 DEmiRNA, and 1211 DEmRNA were obtained, and 96 circRNA-miRNA pairs and 139 miRNA-mRNA pairs were constructed by prediction. The ceRNA regulatory network (circRNA-miRNA-mRNA) was constructed, which included 42 circRNA, 36miRNA, and 78 mRNA. GO function annotation showed genes were mainly enriched in receptor activity activated by transforming growth factor beta (TGF-beta) and in the regulation of epithelial cell apoptosis. KEGG analysis showed genes were mainly enriched in the TGF-beta signaling, PI3K-Akt signaling, and Wnt signaling pathways. Four genes associated with survival and prognosis of breast cancer were obtained by survival analysis, the prognostic sub-network included 4 circRNA, 4 miRNA, and 4 mRNA. BCIP analysis and qRT-PCR verification confirmed that relative mRNA expression levels were consistent with those in the GEO database. CONCLUSION: A circRNA-related ceRNA regulatory network was constructed for breast cancer in this study and key genes affecting pathogenesis and progression were identified. These findings may help better understand and further explore the molecular mechanisms that affect the progression and pathogenesis of breast cancer.
Subject(s)
Breast Neoplasms/genetics , RNA, Circular/genetics , Computational Biology/methods , Databases, Genetic , Early Detection of Cancer/methods , Female , Gene Expression/genetics , Gene Expression Regulation, Neoplastic/genetics , Gene Regulatory Networks/genetics , Humans , Mass Screening , MicroRNAs/genetics , Protein Interaction Maps/genetics , RNA, Circular/analysis , RNA, Messenger/genetics , Signal Transduction/genetics , Transcriptome/geneticsABSTRACT
Here we describe a new integrative approach for accurate annotation and quantification of circRNAs named Short Read circRNA Pipeline (SRCP). Our strategy involves two steps: annotation of validated circRNAs followed by a quantification step. We show that SRCP is more sensitive than other individual pipelines and allows for more comprehensive quantification of a larger number of differentially expressed circRNAs. To facilitate the use of SRCP, we generate a comprehensive collection of validated circRNAs in five different organisms, including humans. We then utilize our approach and identify a subset of circRNAs bound to the miRNA-effector protein AGO2 in human brain samples.
Subject(s)
Molecular Sequence Annotation , RNA, Circular/analysis , Software , Animals , Argonaute Proteins/metabolism , Brain/metabolism , Databases, Nucleic Acid , Exoribonucleases , Genomics , Humans , Mice , RNA, Circular/genetics , RNA, Circular/metabolism , RNA-Seq , RatsABSTRACT
PURPOSE: To detect the expression level of hsa_circ_0005721 in osteosarcoma specimen and plasma of osteosarcoma patients, and to analyze the clinical significance of hsa_circ_0005721 as a diagnostic marker for osteosarcoma. METHODS: Expression levels of hsa_circ_0005721 in osteosarcoma specimen and osteosarcoma cell lines were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between hsa_circ_0005721 expression difference and overall survival in osteosarcoma was analyzed by Kaplan-Meier method. Expression level of hsa_circ_0005721 was stably downregulated in U-2OS and HOS cells by shRNA transfection. Proliferative potential in osteosarcoma cells regulated by hsa_circ_0005721 was assessed by colony formation and 5-Ethynyl-2'- deoxyuridine (EdU) assay. Differentially expressed hsa_circ_0005721 in the plasma of healthy controls, benign bone tumor patients and osteosarcoma patients was determined by qRT-PCR. The diagnostic capacity of hsa_circ_0005721 in osteosarcoma was examined by receiver operating characteristic (ROC) curves. RESULTS: hsa_circ_0005721 was upregulated in osteosarcoma specimen and osteosarcoma cell lines. High level of hsa_circ_0005721 predicted poor prognosis in osteosarcoma patients. In vitro experiments showed that knockdown of hsa_circ_0005721 suppressed proliferative ability in osteosarcoma cells. Compared with that in healthy controls and benign bone tumor patients, plasma level of hsa_circ_0005721 was higher in osteosarcoma patients. ROC curves demonstrated the diagnostic potential of hsa_circ_0005721 in osteosarcoma. CONCLUSIONS: hsa_circ_0005721 is upregulated in osteosarcoma samples, which acts as an oncogene responsible for aggravating the progression. hsa_circ_0005721 can be a promising diagnostic marker for osteosarcoma.
Subject(s)
Biomarkers, Tumor/analysis , Bone Neoplasms/blood , Bone Neoplasms/chemistry , Osteosarcoma/blood , Osteosarcoma/chemistry , RNA, Circular/analysis , Biomarkers, Tumor/genetics , Bone Neoplasms/diagnosis , Humans , Osteosarcoma/diagnosis , RNA, Circular/genetics , Tumor Cells, CulturedABSTRACT
Despite significant advances in cancer therapy strategies, breast cancer is one of the most common and lethal malignancies worldwide. Characterization of a new class of RNAs using next-generation sequencing opened new doors toward uncovering etiopathogenesis mechanisms of breast cancer as well as prognostic and diagnostic biomarkers. Circular RNAs (circRNAs) are a novel class of RNA with covalently closed and highly stable structures generated primarily from the back-splicing of precursor mRNAs. Although circRNAs exert their function through various mechanisms, acting as a sponge for miRNAs is their primary mechanism of function. Furthermore, growing evidence has shown that aberrant expression of circRNAs is involved in the various hallmarks of cancers. This paper reviews the biogenesis, characteristics, and mechanism of functions of circRNAs and their deregulation in various cancers. Finally, we focused on the circRNAs roles as a sponge for miRNAs in the development, metastasis, angiogenesis, drug resistance, apoptosis, and immune responses of breast cancer.
Subject(s)
Biomarkers/metabolism , Breast Neoplasms/genetics , Gene Expression Regulation, Neoplastic , MicroRNAs/metabolism , RNA, Circular/metabolism , Biomarkers/analysis , Breast Neoplasms/diagnosis , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Female , Humans , Prognosis , RNA, Circular/analysis , Survival RateABSTRACT
Circular RNAs (circRNAs) are an emerging class of regulatory RNAs with a covalently closed-loop structure formed during pre-mRNA splicing. Recent advances in high-throughput RNA sequencing and circRNA-specific computational tools have driven the development of novel approaches to their identification and functional characterization. CircRNAs are stable, developmentally regulated, and show tissue- and cell-type-specific expression across different taxonomic groups. They play a crucial role in regulating various biological processes at post-transcriptional and translational levels. However, the involvement of circRNAs in fish immunity has only recently been recognized. There is also broad evidence in mammals that the timely expression of circRNAs in muscle plays an essential role in growth regulation but our understanding of their expression and function in teleosts is still very limited. Here, we discuss the available knowledge about circRNAs and their role in growth and immunity in vertebrates from a comparative perspective, with emphasis on cultured teleost fish. We expect that the interest in teleost circRNAs will increase substantially soon, and we propose that they may be used as biomarkers for selective breeding of farmed fish, thus contributing to the sustainability of the aquaculture sector.
Subject(s)
Aquaculture/methods , Fishes/genetics , RNA, Circular/genetics , Animals , Biomarkers , Fishes/growth & development , High-Throughput Nucleotide Sequencing/methods , Immunity/genetics , RNA/genetics , RNA Splicing/genetics , RNA, Circular/analysisABSTRACT
BACKGROUND: Malignant tumor is one of the most serious diseases endangering human health. Circular RNAs play an important role in the tumorigenesis and progression of various malignant tumors. Although various studies have investigated the biological function of circular RNA circSMARCA5 in malignant tumors, the prognostic value of circSMARCA5 in malignant tumor patients has not been systematically analyzed. METHODS: Relevant studies were obtained from the PubMed and Web of Science database. The quality of the enrolled studies was evaluated using the Newcastle-Ottawa Scale quality assessment system. Survival features and clinicopathological features were assessed using pooled hazard ratios and odds ratios with 95% confidence intervals, respectively. RESULTS: Overall, 7 relevant publications were enrolled in the meta-analysis. CircSMARCA5 expression was significantly correlated with better OS (HR = 0.51, 95%CI 0.41-0.65) or DFS/RFS/PFS (HR = 0.56, 95%CI 0.43-0.73) in malignant tumors. In the pooled analyses of clinicopathological characteristics, malignant tumors with higher circSMARCA5 were better differentiated (OR = 0.41, 95%CI 0.19-0.88). CircSMARCA5 expression was correlated with less advanced TNM stage (OR = 0.33, 95%CI 0.19-0.55). Moreover, malignant tumors with higher circSMARCA5 expression have less advanced lymph node metastasis (OR = 0.26, 95%CI 0.08-0.79). CONCLUSION: These results indicated that circSMARCA5 was a promising biomarker in malignant tumors, which may potentially facilitate clinical decisions in the future.
Subject(s)
Biomarkers, Tumor/metabolism , Gene Expression Regulation, Neoplastic , Neoplasms/mortality , RNA, Circular/metabolism , Biomarkers, Tumor/analysis , Carcinogenesis/genetics , Cell Differentiation/genetics , Humans , Lymphatic Metastasis/genetics , Neoplasms/genetics , Neoplasms/pathology , Neoplasms/therapy , Prognosis , RNA, Circular/analysisABSTRACT
OBJECTIVES: Circular RNAs (circRNAs) are noncoding RNAs that compete against other endogenous RNA species, such as microRNAs, and have been implicated in many diseases. In this study, we investigated the role of a new circRNA (circSLC7A2) in osteoarthritis (OA). MATERIALS AND METHODS: The relative expression of circSLC7A2 was significantly lower in OA tissues than it was in matched controls, as shown by real-time quantitative polymerase chain reaction (RT-qPCR). Western blotting, RT-qPCR and immunofluorescence experiments were employed to evaluate the roles of circSLC7A2, miR-4498 and TIMP3. The in vivo role and mechanism of circSLC7A2 were also conformed in a mouse model. RESULTS: circSLC7A2 was decreased in OA model and the circularization of circSLC7A2 was regulated by FUS. Loss of circSLC7A2 reduced the sponge of miR-4498 and further inhibited the expression of TIMP3, subsequently leading to an inflammatory response. We further determined that miR-4498 inhibitor reversed circSLC7A2-knockdown-induced OA phenotypes. Intra-articular injection of circSLC7A2 alleviated in vivo OA progression in a mouse model of anterior cruciate ligament transection (ACLT). CONCLUSIONS: The circSLC7A2/miR-4498/TIMP3 axis of chondrocytes catabolism and anabolism plays a critical role in OA development. Our results suggest that circSLC7A2 may serve as a new therapeutic target for osteoarthritis.
Subject(s)
Osteoarthritis/genetics , RNA, Circular/genetics , Tissue Inhibitor of Metalloproteinase-3/genetics , Animals , Apoptosis , Cartilage, Articular/metabolism , Cartilage, Articular/pathology , Down-Regulation , Gene Expression Regulation , Humans , Mice , MicroRNAs/genetics , Osteoarthritis/pathology , RNA, Circular/analysis , Tissue Inhibitor of Metalloproteinase-3/analysisABSTRACT
Analysis of circular RNA (circRNA) expression from RNA-Seq data can be performed with different algorithms and analysis pipelines, tools allowing the extraction of heterogeneous information on the expression of this novel class of RNAs. Computational pipelines were developed to facilitate the analysis of circRNA expression by leveraging different public tools in easy-to-use pipelines. This chapter describes the complete workflow for a computationally reproducible analysis of circRNA expression starting for a public RNA-Seq experiment. The main steps of circRNA prediction, annotation, classification, sequence reconstruction, quantification, and differential expression are illustrated.