Journal: BioMed Research International

Article Title: Circular RNA Profiling and Bioinformatic Modeling Identify Its Regulatory Role in Hepatic Steatosis

doi: 10.1155/2017/5936171

Figure Lengend Snippet: circRNA profiles differentiate the normal group from model group. (a) Principal component analysis (PCA) shows the difference between normal and model groups. (b) Scatter plots assess the variation of circRNA expression between two groups. The values plotted on x and y axes are the normalized signal values of each group (log2 scaled). Dots above the top green line and below the bottom green line represent differentially expressed circRNAs. (c, d) Unsupervised hierarchical clustering (c) and volcano plot (d) demonstrate the differential expression of circRNAs during hepatic steatosis. Both downregulated (green) and upregulated (red) circRNAs were visualized in the cluster. In similar, the red points in volcano plot represent the differentially expressed circRNAs with statistical significance. (e, f) Validation of the upregulated (e) and downregulated circRNAs (f) using QPCR. (g) The results of QPCR exhibit well consistence with those of microarray. Pairwise scatter plots reflect the fold changes (log2 transformed) of both microarray (horizontal axis) and the QPCR (vertical axis). The R stands for linear correlation coefficient. The presented results are expressed as means ± SD. ∗∗ P < 0.01.

Article Snippet: To remove linear RNAs and enrich circRNA, total RNA from each sample was treated with Rnase R. circRNA was then transcribed into fluorescent cRNA by random primer according to the Arraystar Super RNA Labeling protocol (Arraystar, Inc., USA) and hybridized onto the Arraystar Human circRNA Arrays V2.0 (Arraystar, Inc., USA) at 65°C for 17 hours.

Techniques: Expressing, Quantitative Proteomics, Biomarker Discovery, Microarray, Transformation Assay

Journal: BioMed Research International

Article Title: Circular RNA Profiling and Bioinformatic Modeling Identify Its Regulatory Role in Hepatic Steatosis

doi: 10.1155/2017/5936171

Figure Lengend Snippet: Predicted targets of top-20 differentially expressed circRNAs.

Article Snippet: To remove linear RNAs and enrich circRNA, total RNA from each sample was treated with Rnase R. circRNA was then transcribed into fluorescent cRNA by random primer according to the Arraystar Super RNA Labeling protocol (Arraystar, Inc., USA) and hybridized onto the Arraystar Human circRNA Arrays V2.0 (Arraystar, Inc., USA) at 65°C for 17 hours.

Techniques:

Journal: BioMed Research International

Article Title: Circular RNA Profiling and Bioinformatic Modeling Identify Its Regulatory Role in Hepatic Steatosis

doi: 10.1155/2017/5936171

Figure Lengend Snippet: Enumeration data of differentially expressed circRNAs and target miRNAs/mRNAs.

Article Snippet: To remove linear RNAs and enrich circRNA, total RNA from each sample was treated with Rnase R. circRNA was then transcribed into fluorescent cRNA by random primer according to the Arraystar Super RNA Labeling protocol (Arraystar, Inc., USA) and hybridized onto the Arraystar Human circRNA Arrays V2.0 (Arraystar, Inc., USA) at 65°C for 17 hours.

Techniques:

Journal: BioMed Research International

Article Title: Circular RNA Profiling and Bioinformatic Modeling Identify Its Regulatory Role in Hepatic Steatosis

doi: 10.1155/2017/5936171

Figure Lengend Snippet: circRNA-miRNA-mRNA regulatory network uncovers the circRNA_021412-miR-1972-LPIN1 signaling underlying circRNAs' effects. (a) The circRNA-miRNA-mRNA network related to transcriptional regulation. Red cycles, violet squares, and blue cycles represent circRNAs, miRNAs, and mRNAs, respectively. The size of each symbol reflects its degree, which is scored by the number of downstream targets. (b) circRNA_021412-miR-1972-LPIN1 signaling within the circRNA-miRNA-mRNA network is recognized to underlie the actions of circRNAs. (c) The circRNA_021412-miR-1972-LPIN1 signaling controls the pathway of lipid degradation via PPAR α -induced ACSLs expression.

Article Snippet: To remove linear RNAs and enrich circRNA, total RNA from each sample was treated with Rnase R. circRNA was then transcribed into fluorescent cRNA by random primer according to the Arraystar Super RNA Labeling protocol (Arraystar, Inc., USA) and hybridized onto the Arraystar Human circRNA Arrays V2.0 (Arraystar, Inc., USA) at 65°C for 17 hours.

Techniques: Expressing

Journal: BioMed Research International

Article Title: Circular RNA Profiling and Bioinformatic Modeling Identify Its Regulatory Role in Hepatic Steatosis

doi: 10.1155/2017/5936171

Figure Lengend Snippet: Diagram of circRNA_021412-based regulation of hepatic steatosis. Arrow, blunt-headed line, and red cross represent effects of activation, inhibition, and blocking, respectively.

Article Snippet: To remove linear RNAs and enrich circRNA, total RNA from each sample was treated with Rnase R. circRNA was then transcribed into fluorescent cRNA by random primer according to the Arraystar Super RNA Labeling protocol (Arraystar, Inc., USA) and hybridized onto the Arraystar Human circRNA Arrays V2.0 (Arraystar, Inc., USA) at 65°C for 17 hours.

Techniques: Activation Assay, Inhibition, Blocking Assay

Journal: Cell Death & Disease

Article Title: Angiotensin II inhibits apoptosis of mouse aortic smooth muscle cells through regulating the circNRG-1/miR-193b-5p/NRG-1 axis

doi: 10.1038/s41419-019-1590-5

Figure Lengend Snippet: a Scatter plots were used to evaluate the difference in the expression of circRNAs between Ang II and control groups. The values plotted on X and Y axes are the averaged normalized signal values of each group (log2 scaled). The circRNAs above the top green line and below the bottom green line indicate >1.5-fold change between the two groups. b Hierarchical clustering analysis showed the differentially expressed circRNAs over 2.0-fold change. Red color indicates high expression level, and blue color indicates low expression level. c Divergent and convergent primers were used to verify whether circNRG-1 was a circRNA. Convergent primers were used to detect NRG-1 mRNA. Divergent primers amplified circNRG-1 in cDNA but not gDNA. GAPDH served as linear control and size marker in base pairs. d Sanger sequencing confirmed head-to-tail junction of circNRG-1. e RNA fluorescence in situ hybridization for circNRG-1 was detected. Nuclei were stained with DAPI. Scale bars = 50 μm. f qRT-PCR detected circNRG-1 expression in MASMCs treated with Ang II (10 −7 M) for the different times. Data represent the means ± SEM of three independent experiments. * P < 0.05, *** P < 0.001 vs . Ang II for 0 h

Article Snippet: Circular RNA expression profiling was performed using Arraystar Mouse circRNA Array V2 analysis (Arraystar, USA).

Techniques: Expressing, Control, Amplification, Marker, Sequencing, Fluorescence, In Situ Hybridization, Staining, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Angiotensin II inhibits apoptosis of mouse aortic smooth muscle cells through regulating the circNRG-1/miR-193b-5p/NRG-1 axis

doi: 10.1038/s41419-019-1590-5

Figure Lengend Snippet: The orange, purple and green nodes represent circRNA, miRNA and mRNA respectively. Markers highlighting staining showed circNRG-1-miR-193b-5p-NRG-1 interactions

Article Snippet: Circular RNA expression profiling was performed using Arraystar Mouse circRNA Array V2 analysis (Arraystar, USA).

Techniques: Staining

Journal: Cell Death & Disease

Article Title: Circ ERCC2 ameliorated intervertebral disc degeneration by regulating mitophagy and apoptosis through miR-182-5p/SIRT1 axis

doi: 10.1038/s41419-019-1978-2

Figure Lengend Snippet: a Volcano plots showed differential expression of circRNAs detected by circRNA microarray in IVDD compared with the control. b Volcano plots showed differential expression of circRNAs in GSE67566. c The 9 downregulated circRNAs in IVDD were identified based on the overlap of circRNA microarray and GSE67566. d Heatmap of 9 circRNAs in circRNA microarray and heatmap of 9 circRNAs in GSE67566. e qRT-PCR analysis confirmed the downregulation of circRNAs in IVDD compared with control. * p < 0.05. f circ ERCC2 is transcribed from 13, 14, and 15 exons of the ERCC2 gene. The expression of circ ERCC2 was lower in NP tissues from IVDD compared with the control detected by FISH. g FISH detection of circ ERCC2 in the cytoplasm of NPCs. In ( f ) and ( g ), blue fluorescence indicated the nucleus and green fluorescence indicated circ ERCC2. Scale bar: 20 μm. h Representative plots of apoptosis detected by flow cytometry. circ ERCC2 inhibited the rate of apoptosis of NPCs. * p < 0.05, ** p < 0.01. i NPCs were treated by TBHP or/and circ ERCC2, and mitophagy and apoptosis related proteins were detected by Western blot analysis

Article Snippet: Identification of differentially expressed circRNAs was performed by overlapping microarray analysis of human circRNAs (Arraystar, CA, USA) and microarray dataset (GSE67566) obtained from Gene Expression Omnibus (GEO) database.

Techniques: Quantitative Proteomics, Microarray, Control, Quantitative RT-PCR, Expressing, Fluorescence, Flow Cytometry, Western Blot

Journal: Medicine

Article Title: Comprehensive Circular RNA Profiling Reveals That hsa_circ_0005075, a New Circular RNA Biomarker, Is Involved in Hepatocellular Crcinoma Development

doi: 10.1097/MD.0000000000003811

Figure Lengend Snippet: Differentially expressed circRNAs between HCC tissues and adjacent nontumorous tissues. The result from unsupervised hierarchical clustering analysis shows distinguishable circRNA expression profiling among samples (H for HCC and N for normal adjacent nontumorous tissues). Each column represents the expression profile of a tissue sample, and each row corresponds to a circRNA. “Red” indicates higher expression level, and “green” indicates lower expression level. HCC = hepatocellular carcinoma.

Article Snippet: And then, the microarray analysis of circRNAs was conducted according to the manufacturer's protocol (Arraystar, Rockville, MD).

Techniques: Expressing

Journal: Medicine

Article Title: Comprehensive Circular RNA Profiling Reveals That hsa_circ_0005075, a New Circular RNA Biomarker, Is Involved in Hepatocellular Crcinoma Development

doi: 10.1097/MD.0000000000003811

Figure Lengend Snippet: The expression levels of candidate circRNAs for validation in HCC tissue and adjacent liver tissue samples. A, Hsa_circ_0005075, hsa_circ_0000520, and hsa_circ_0066444 expression levels were examined in 60 paired tissue samples by qRT-PCR; only hsa_circ_0005075 was significantly differentially expressed between the 2 groups ( P < 0 .001). B, The expression levels of hsa_circ_0005075 in each patient with comparison between cancer (HCC) and adjacent normal tissues (n = 30). C, The expression levels of hsa_circ_0005075 in the HCC group are significantly higher than those in corresponding nontumorous tissues ( P < 0 .001).

Article Snippet: And then, the microarray analysis of circRNAs was conducted according to the manufacturer's protocol (Arraystar, Rockville, MD).

Techniques: Expressing, Biomarker Discovery, Quantitative RT-PCR, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Comprehensive analysis of circular RNAs in pathological states: biogenesis, cellular regulation, and therapeutic relevance

doi: 10.1007/s00018-019-03016-5

Figure Lengend Snippet: The biogenesis of circRNAs. a Through the pre-mRNA canonical splicing, the mature mRNA is generated. The alternative splicing pathways are: exon skipping and back-splicing. b The circularized transcript can contain only the exon part and it is named exonic circular RNA (ecircRNA/circRNA). c, d The flanking introns can form direct base-pairing (c) or RNA-binding protein (RNP)-mediated intron–intron binding (d). The resulted circular transcript can be an exon–intron circular RNA (EIcircRNA) or the introns are further removed and the two- or multiple exons containing ecircRNA are generated. The EIcircRNA can regulate its own transcription by interacting with U1 snRNA and RNA Pol II. e The intron can form a lariat during canonical or alternative splicing. The lariat may become more stable and circular thus giving rise to the intronic circular RNA (circRNA). f In bacterial cells, as well as in eukaryotic cells, it was proven that the primary tRNA has introns that are spliced and circularized into tricRNA. Their function still remains to be studied

Article Snippet: Arraystar is the most common circRNAs microarray platform, based on an adapted protocol that uses RNase R treatment and a specific circular junction probe design to achieve a high sensitivity and specificity through enriched circRNAs’ fraction [ 121 ].

Techniques: Generated, Alternative Splicing, RNA Binding Assay, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Comprehensive analysis of circular RNAs in pathological states: biogenesis, cellular regulation, and therapeutic relevance

doi: 10.1007/s00018-019-03016-5

Figure Lengend Snippet: Relevant examples of circRNAs implication in cancer

Article Snippet: Arraystar is the most common circRNAs microarray platform, based on an adapted protocol that uses RNase R treatment and a specific circular junction probe design to achieve a high sensitivity and specificity through enriched circRNAs’ fraction [ 121 ].

Techniques: Inhibition, Expressing, Biomarker Discovery, In Vitro, Knockdown, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Comprehensive analysis of circular RNAs in pathological states: biogenesis, cellular regulation, and therapeutic relevance

doi: 10.1007/s00018-019-03016-5

Figure Lengend Snippet: Relevant examples of circRNAs’ implication in cardiovascular disease, diabetes, aging, and regenerative medicine

Article Snippet: Arraystar is the most common circRNAs microarray platform, based on an adapted protocol that uses RNase R treatment and a specific circular junction probe design to achieve a high sensitivity and specificity through enriched circRNAs’ fraction [ 121 ].

Techniques: Expressing, Migration, Alternative Splicing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Comprehensive analysis of circular RNAs in pathological states: biogenesis, cellular regulation, and therapeutic relevance

doi: 10.1007/s00018-019-03016-5

Figure Lengend Snippet: Summarisation of the principle, advantage, and disadvantages of the main detection methods used for quantification of circRNAs

Article Snippet: Arraystar is the most common circRNAs microarray platform, based on an adapted protocol that uses RNase R treatment and a specific circular junction probe design to achieve a high sensitivity and specificity through enriched circRNAs’ fraction [ 121 ].

Techniques: Next-Generation Sequencing, Sequencing, Genome Wide, Microarray, Purification, Hybridization, cDNA Synthesis, Amplification, Biomarker Discovery, Northern Blot, Agarose Gel Electrophoresis, Immunoprecipitation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Comprehensive analysis of circular RNAs in pathological states: biogenesis, cellular regulation, and therapeutic relevance

doi: 10.1007/s00018-019-03016-5

Figure Lengend Snippet: Examples of databases and software for circRNAs evaluation

Article Snippet: Arraystar is the most common circRNAs microarray platform, based on an adapted protocol that uses RNase R treatment and a specific circular junction probe design to achieve a high sensitivity and specificity through enriched circRNAs’ fraction [ 121 ].

Techniques: Software, Expressing, Genome Wide, Modification

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Comprehensive analysis of circular RNAs in pathological states: biogenesis, cellular regulation, and therapeutic relevance

doi: 10.1007/s00018-019-03016-5

Figure Lengend Snippet: Therapeutic value of synthetic circRNAs. a The synthetic circRNAs can be design in such a way that it will be able to target multiple miRNAs with consequences toward impairment of multiple oncogenic pathways. Synthetic circRNA can be delivered as a single agent. Inside the cell, this circRNA will sponge multiple microRNAs; b a second option consists in synthetic circRNAs able to target multiple proteins leading to the impairment of multiple oncogenic pathways. Once delivered inside the cell, the oncogenic proteins will be sponged and their activity suppressed; c both types of circRNAs can be loaded in tumor cell exosomes which will enhance their targeting efficiency and amount that can be delivered

Article Snippet: Arraystar is the most common circRNAs microarray platform, based on an adapted protocol that uses RNase R treatment and a specific circular junction probe design to achieve a high sensitivity and specificity through enriched circRNAs’ fraction [ 121 ].

Techniques: Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Comprehensive analysis of circular RNAs in pathological states: biogenesis, cellular regulation, and therapeutic relevance

doi: 10.1007/s00018-019-03016-5

Figure Lengend Snippet: The major strategies for circularRNA therapy. a A tumor suppressor circRNA inside the cell will sponge oncomiRs, resulting in the upregulation of tumor suppressor mRNAs. b In the intracellular environment, siRNA can target and repress the oncogenic circRNA and the upregulation of tumor suppressor mRNA. c Inside the cell, the number of tumor suppressor circRNAs is restored and these can directly inhibit the oncogenic mRNAs. d circRNAs can also function as miRNA delivery systems, where tumor suppressor miRNAs can further repress the oncogenic mRNA. All of the above-mentioned circRNA therapeutic strategies result in the mRNA-mediated tumor suppression

Article Snippet: Arraystar is the most common circRNAs microarray platform, based on an adapted protocol that uses RNase R treatment and a specific circular junction probe design to achieve a high sensitivity and specificity through enriched circRNAs’ fraction [ 121 ].

Techniques: