CircRNA sequencing in peripheral blood mononuclear cells from Ankylosing Spondylitis patients and healthy controls
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE178408 | GEO | 2022/01/05
REPOSITORIES: GEO
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Project description:Purpose: With the advent of human genome sequencing project, circRNAs attracted widespread attention in cancer research due to its stable ring structure. Our aim was to identify differentially expressed circRNAs in GC and explore their potential roles in GC diagnosis and treatment. Methods: Total RNA from tissues was isolated by Hipure Total RNA Mini Kit (Magen, Germany). Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, California) was used for RNA concentration measurement while Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA) for RNA integrity estimation. A RIN value over 7.0 was considered eligible. RNA-seq library was prepared with approximately 2μg of total RNA using KAPA RNA HyperPrep Kit with RiboErase (HMR) for Illumina® (Kapa Biosystems, Inc., Woburn, MA). Briefly, total RNA was incubated at 37 °C for 30 min with 10 units RNase R (Epicentre Technologies, Madison, USA) after removing ribosomal RNA. Next, the ribominus RNase R (+) RNAs was fragmented and then first strand and directional second strand synthesis were performed. Then the A tailing and adapter ligation were performed with the purified cDNA. Finally, the purified, adapter-ligated DNA was amplified. Each library was diluted to 10 nM and pooled equimolar prior to clustering. Paired-End (PE150) sequencing was performed on all samples. Results: A total of 25,303 circRNA targets, including 20,036 known circRNAs and 5,267 uncharacterized circRNAs, were detected and defined. Among them, there were 2,007 circRNAs with statistically significant differences in expression in GC tissues, in which fold changes>2.0 and P<0.05 were identified.
2019-06-16 | GSE131414 | GEO
Project description:Circular RNAs (circRNAs), a noncoding RNA class originating from alternative splicing, are highly abundant in neural tissues and were shown to regulate gene expression e.g. by interacting with microRNAs and RNA-binding proteins. Neuroblastoma is an embryonal neoplasia, which arises from undifferentiated neural crest cells. Here, we aimed to explore, whether circRNAs influence the pathogenesis of high-risk neuroblastoma. We performed whole-transcriptome sequencing of 104 primary neuroblastoma samples of all risk-groups and identified 5,203 unique circRNAs involving 2,302 genes. Candidate circRNA expression did not correlate with host gene expression, indicating independent regulatory mechanisms. circRNAs were significantly downregulated in the MYCN-amplified high-risk tumors. These findings were independently reproduced in a tetracycline-inducible MYCN-overexpression system based on a non MYCN-amplified neuroblastoma cell line, suggesting that MYCN drives this global circRNA repression. We identified the RNA helicase DHX9 as a mediator of this global suppressive effect of MYCN on circRNAs. Comparing our RNA sequencing data with other cancers and controls revealed a circRNA subset specifically upregulated in neuroblastoma that included a circRNA derived from the ARID1A tumor suppressor gene. Specific circARID1A knockdown resulted in reduced proliferation, cell numbers and viability, prompted apoptosis and induced a differentiated phenotype. Neither knockdown, nor overexpression of circARID1A influenced ARID1A mRNA and protein levels significantly. To dissect the potential mode of function, we performed a pulldown assay with subsequent mass spectrometry. We identified the RNA-binding protein KHSRP as an interaction partner that participates in the mechanism of action of circARID1A. In summary, this study highlights an important role of circRNAs in neuroblastoma biology and may establish this RNA class as a future therapeutic target and biomarker.
2023-04-06 | PXD026053 | Pride
Project description:Objective: Bladder outlet obstruction (BOO) is a common urologic disease associated with poorly understood molecular mechanisms. This study aimed to investigate the possible involvements of circRNAs (circular RNAs) and circRNA-encoded proteins in BOO development. Methods: The rat BOO model was established by the partial bladder outlet obstruction surgery. Differential expression of circRNA and protein profiles were characterized by deep RNA sequencing and iTRAQ quantitative proteomics respectively. Novel proteins encoded by circRNAs were predicted through ORF (open reading frame) selection using the GETORF software and verified by the mass spectrometry in proteomics, combined with the validation of their expressional alterations by quantitative RT-PCR. Results: Totally 3051 circRNAs were differentially expressed in bladder tissues of rat BOO model with widespread genomic distributions, including 1414 up-regulated and 1637 down-regulated circRNAs. Our following quantitative proteomics revealed significant changes of 85 proteins in rat BOO model, which were enriched in multiple biological processes and signaling pathways such as the PPAR and Wnt pathways. Among them, 21 differentially expressed proteins were predicted to be encoded by circRNAs and showed consistent circRNA and protein levels in rat BOO model. The expression of five protein-encoding circRNA were further validated by quantitative RT-PCR and mass spectrometry. Conclusion: The circRNA and protein profiles were substantially altered in rat BOO model, with great expressional changes of circRNA-encoded novel proteins.
2022-02-22 | PXD029337 | Pride
Project description:Circular RNAs (circRNAs) are a large class of animal RNAs. To investigate possible circRNA functions, it is important to understand circRNA biogenesis. Besides human Alu repeats, sequence features that promote exon circularization are largely unknown. We experimentally identified new circRNAs in C. elegans. Reverse complementary sequences between introns bracketing circRNAs were significantly enriched compared to linear controls. By scoring the presence of reverse complementary sequences in human introns we predicted and experimentally validated novel circRNAs. We show that introns bracketing circRNAs are highly enriched in RNA editing or hyper-editing events. Knockdown of the double-strand RNA editing ADAR1 enzyme significantly and specifically up-regulated circRNA expression. Together, our data support a model of animal circRNA biogenesis in which competing RNA:RNA interactions of introns form larger structures which promote circularization of embedded exons, while ADAR1 antagonizes circRNA expression by melting stems within these interactions. Thus, we assign a new function to ADAR1. Examination of 12 samples in different stages of C.elegans development.
2015-01-06 | E-GEOD-63823 | biostudies-arrayexpress
Project description:Circular RNAs (circRNAs), formed by the atypical head-to-tail splicing of exons, have re-emerged as a potentially interesting RNA species given recent reports of a surprising diversity and abundance of circRNA in organisms ranging from worm to human. Here, using deep RNA sequencing, we profiled different RNA species in mouse and observed that circRNAs are significantly enriched in neural tissue, relative to other tissues. Using PacBio sequencing, we determined, for the first time, the circular structure of this population of circRNAs as well as their full-length sequences. We discovered that a disproportionate fraction of the brain circRNA population is derived from host genes that code for synaptic proteins. Moreover, based on the separate profiling of the RNAs localized in neuronal cell bodies and neuropil (enriched in axons and dendrites), we found that, on average, circular RNAs are more enriched in the neuropil than their host gene mRNA isoforms. Using high resolution in situ hybridization we, for the first time, directly visualized circRNA punctae in the dendrites of neurons. The host gene origin and location of the circRNA in neurons suggest the possibility that circRNAs might participate in the regulation of synaptic function and plasticity. Consistent with this idea, we observed via profiling at different developmental stages, that the abundance of many circular RNAs changes abruptly at a time corresponding to synaptogenesis. In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibit significant up or down-regulation. These data indicate that brain circRNAs are positioned to respond to and regulate synaptic function. Circular RNA profiling in 13 different samples in mice and four samples in rat, using Illumina sequencing
2015-02-25 | E-GEOD-61991 | biostudies-arrayexpress
Project description:The human genome encodes tens of thousands circular RNAs (circRNAs) whose levels correlate with many disease states. While studies have focused on the non-coding functions of circRNAs, emerging evidence suggests that a handful of circRNAs encode proteins. Translation canonically starts by recognition of mRNA 5’cap and scanning to the start codon; how circRNA translation initiates remains unclear. Here, we developed a high-throughput screen to systematically identify and quantify RNA sequences that can direct circRNA translation. We identify and validate over 17,000 circRNA internal ribosome entry sites (IRES) and reveal that 18S rRNA complementarity and a structured RNA element on the IRES are important for facilitating circRNA cap-independent translation. With genomic and peptidomic analyses of the IRES, we identified nearly 1,000 putative endogenous protein-coding circRNAs and hundreds of translational units encoded by these circRNAs. We further characterized circFGFR1p, a protein encoded by circFGFR1, functions as a negative regulator of FGFR1 to suppress cell growth under stress conditions. The circRNA proteome may be important links among circRNA, biological control, and disease.
2021-08-26 | PXD025203 | Pride
Project description:The human genome encodes tens of thousands circular RNAs (circRNAs) whose levels correlate with many disease states. While studies have focused on the non-coding functions of circRNAs, emerging evidence suggests that a handful of circRNAs encode proteins. Translation canonically starts by recognition of mRNA 5’cap and scanning to the start codon; how circRNA translation initiates remains unclear. Here, we developed a high-throughput screen to systematically identify and quantify RNA sequences that can direct circRNA translation. We identify and validate over 17,000 circRNA internal ribosome entry sites (IRES) and reveal that 18S rRNA complementarity and a structured RNA element on the IRES are important for facilitating circRNA cap-independent translation. With genomic and peptidomic analyses of the IRES, we identified nearly 1,000 putative endogenous protein-coding circRNAs and hundreds of translational units encoded by these circRNAs. We further characterized circFGFR1p, a protein encoded by circFGFR1, functions as a negative regulator of FGFR1 to suppress cell growth under stress conditions. The circRNA proteome may be important links among circRNA, biological control, and disease.
2021-08-25 | PXD025233 | Pride
Project description:The human genome encodes tens of thousands circular RNAs (circRNAs) whose levels correlate with many disease states. While studies have focused on the non-coding functions of circRNAs, emerging evidence suggests that a handful of circRNAs encode proteins. Translation canonically starts by recognition of mRNA 5’cap and scanning to the start codon; how circRNA translation initiates remains unclear. Here, we developed a high-throughput screen to systematically identify and quantify RNA sequences that can direct circRNA translation. We identify and validate over 17,000 circRNA internal ribosome entry sites (IRES) and reveal that 18S rRNA complementarity and a structured RNA element on the IRES are important for facilitating circRNA cap-independent translation. With genomic and peptidomic analyses of the IRES, we identified nearly 1,000 putative endogenous protein-coding circRNAs and hundreds of translational units encoded by these circRNAs. We further characterized circFGFR1p, a protein encoded by circFGFR1, functions as a negative regulator of FGFR1 to suppress cell growth under stress conditions. The circRNA proteome may be important links among circRNA, biological control, and disease.
2021-08-23 | PXD024993 | Pride
Project description:The human genome encodes tens of thousands circular RNAs (circRNAs) whose levels correlate with many disease states. While studies have focused on the non-coding functions of circRNAs, emerging evidence suggests that a handful of circRNAs encode proteins. Translation canonically starts by recognition of mRNA 5’cap and scanning to the start codon; how circRNA translation initiates remains unclear. Here, we developed a high-throughput screen to systematically identify and quantify RNA sequences that can direct circRNA translation. We identify and validate over 17,000 circRNA internal ribosome entry sites (IRES) and reveal that 18S rRNA complementarity and a structured RNA element on the IRES are important for facilitating circRNA cap-independent translation. With genomic and peptidomic analyses of the IRES, we identified nearly 1,000 putative endogenous protein-coding circRNAs and hundreds of translational units encoded by these circRNAs. We further characterized circFGFR1p, a protein encoded by circFGFR1, functions as a negative regulator of FGFR1 to suppress cell growth under stress conditions. The circRNA proteome may be important links among circRNA, biological control, and disease.
2021-08-23 | PXD025235 | Pride
Project description:The main aim of the study was to compare genome-wide gene expression profiles obtained from the two widely used commercially available whole blood RNA collection systems - PAXgeneTM and TempusTM tubes. Comparisons of present call rates, variances, correlations and influence of globin reduction across the two collection systems was performed using in vivo glucocorticoid stimulation in 24 peripheral blood samples from three individuals. Three healthy male volunteers aged 30-35 years with body mass index of 20-25 were enrolled in the study.1.5 mg dexamethasone stimulated peripheral blood samples were drawn 3 hours later. For each sample, duplicate measures of 2.5 ml and 3ml blood was collected in PAXgeneTM tubes (PreAnalytiX GmbH, Hombrechtikon, Switzerland) and TempusTM tubes (Applied Biosystems, Foster City, CA, USA) respectively. The PAXgeneTM and TempusTM tubes were stored for 2.5 hours at room temperature and then transferred to -20°C. Due to the lower RNA yield from the PAXgeneTM tubes, RNA from two PAXgeneTM tubes was pooled together for the experiment . Total RNA was isolated from whole blood stored in PAXgeneTM and TempusTM tubes according to the respective manufacturer's instructions. The PAXgeneTM samples were processed using the PAXgeneTM Blood RNA Kit based on the Quiagen method for column purification of nucleic acids (PreAnalytiX GmbH, Hombrechtikon, Switzerland, catalogue number 762174). The TempusTM samples were isolated using the TempusTM Spin RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA, catalogue number 4380204). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The concentration and purity of total RNA was independently assessed by 260/280 UV absorption ratios, respectively (Nanophotometer, Implen, Munich, Germany). RNA samples were divided into non-globin reduced and globin reduced groups. The GLOBINclearTM-Human Kit (Ambion, Inc., Texas, USA, catalogue number #AM1980) was used to remove globin mRNA. Sample amplification and labeling for both globin-reduced and non-reduced samples was performed using the IlluminaR TotalPrep RNA Amplification Kit (Ambion, Inc., Texas, USA, catalogue number AMIL1791).
2012-01-15 | GSE34039 | GEO