Journal: Cell & Bioscience

Article Title: Circular RNA expression profile in blood according to ischemic stroke etiology

doi: 10.1186/s13578-020-00394-3

Figure Lengend Snippet: Distribution by chromosomes ( a ) and chromosome’s region ( b ). Differentially expressed circRNA distribution in chromosomes and chromosome´s region (exonic, intronic, antisense, sense overlapping and intergenic) for each comparison

Article Snippet: To this end, we used Arraystar Human circRNA Array (8 × 15 K, Arraystar), which surveys up to 13,617 probes to identify human circRNAs, in 30 acute stroke patients with different etiologies by TOAST classification [ ].

Techniques:

Journal: Cell & Bioscience

Article Title: Circular RNA expression profile in blood according to ischemic stroke etiology

doi: 10.1186/s13578-020-00394-3

Figure Lengend Snippet: Deregulated circRNA chosen for qPCR validation

Article Snippet: To this end, we used Arraystar Human circRNA Array (8 × 15 K, Arraystar), which surveys up to 13,617 probes to identify human circRNAs, in 30 acute stroke patients with different etiologies by TOAST classification [ ].

Techniques:

Journal: Cell & Bioscience

Article Title: Circular RNA expression profile in blood according to ischemic stroke etiology

doi: 10.1186/s13578-020-00394-3

Figure Lengend Snippet: RT-qPCR validation of hsa_circRNA_102488. a Schematic representation of the UBA52 gene, origin of hsa_circRNA_102488. The graph shows the map of hsa_circRNA_102488 which is originated from exons 2 and 3 of its host mRNA (NCBI RefSeq track shown in the UCSC Genome Browser). b hsa_circRNA_102488 backsplicing. The Sanger-Sequencing electropherogram specifies the sequence of the backspliced junction that results in the circRNA formation. c – e hsa_circRNA_102488 and UBA52 mRNA expression levels between atherotrombotic vs cardioembolic samples. Percentage of hsa_circRNA_102488 expression relative to GAPDH mRNA ( c ) or UBA52 mRNA ( d ) is decreased in atherotrombotic with respect to cardioembolic stroke patients. However, we found similar expression levels of UBA52 mRNA in atherotrombotic and cardioembolic samples groups ( e ). Error bars: 95% CI. **p-value < 0.01; ***p-value < 0.001

Article Snippet: To this end, we used Arraystar Human circRNA Array (8 × 15 K, Arraystar), which surveys up to 13,617 probes to identify human circRNAs, in 30 acute stroke patients with different etiologies by TOAST classification [ ].

Techniques: Quantitative RT-PCR, Sequencing, Expressing

Journal: Cell & Bioscience

Article Title: Circular RNA expression profile in blood according to ischemic stroke etiology

doi: 10.1186/s13578-020-00394-3

Figure Lengend Snippet: GO analysis by mirPath v.3. The image shows the heatmaps corresponding to the analysis performed with the overrepresented miRNAs in the three comparisons (atherotrombotic-cardioembolic-undetermined) by TargetScan ( a ) and the analysis involving the target miRNAs for hsa_circRNA_102488 in accordance with molecular function ( b , by Tarbase) or biological process ( c , by microT-CDS)

Article Snippet: To this end, we used Arraystar Human circRNA Array (8 × 15 K, Arraystar), which surveys up to 13,617 probes to identify human circRNAs, in 30 acute stroke patients with different etiologies by TOAST classification [ ].

Techniques:

Journal: Cell & Bioscience

Article Title: Circular RNA expression profile in blood according to ischemic stroke etiology

doi: 10.1186/s13578-020-00394-3

Figure Lengend Snippet: hsa_circRNA_102488 impact pathways. The cartoon shows miRNAs (in blue) sharing a target site with hsa_circRNA_102488 (in black) and the pathways in which they converge (in dark orange), within those associated with the overrepresented miRNAs in the three comparisons (in light orange). RBPs sites matching to hsa_circRNA_102488 clustered around AGO2 (in dark green) and RBPs sites that matched to its flanking regions clustered around FUS (in light green). ARVC: Arrhythmogenic Right Ventricular Cardiomyopathy; HCM: Hypertrophic Cardiomyopathy

Article Snippet: To this end, we used Arraystar Human circRNA Array (8 × 15 K, Arraystar), which surveys up to 13,617 probes to identify human circRNAs, in 30 acute stroke patients with different etiologies by TOAST classification [ ].

Techniques:

Journal: Scientific Reports

Article Title: A risk progression breast epithelial 3D culture model reveals Cx43/hsa_circ_0077755/miR-182 as a biomarker axis for heightened risk of breast cancer initiation

doi: 10.1038/s41598-021-82057-y

Figure Lengend Snippet: A graphical abstract summarizing the methodology used to select Cx43/has_circ_0077755/miR-182 as the only validated risk-assessment axis for breast cancer initiation. Using the circRNA microarrays and miRNA sequencing results of Cx43-KO-S1 compared to S1 cells and focusing mainly only on Cx43 loss (and hence epithelial polarity loss) and on the sponging activity of circRNAs to miRNAs, three axes were predicted for breast cancer risk-assessment. After using a validation early-stage young breast cancer patient cohort as published in Nassar et al. , the list was narrowed down to only Cx43/has_circ_0077755/miR-182 axis. MREs refer to miRNA response elements, predicted to be “sponged” by the significant circRNAs based on Arraystar's miRNA target prediction software , .

Article Snippet: To identify circRNA expression profile specific to the loss of Cx43, microarrays for circRNAs (Arraystar Human circRNA Array V2) were performed.

Techniques: Sequencing, Activity Assay, Biomarker Discovery, Software

Journal: Scientific Reports

Article Title: A risk progression breast epithelial 3D culture model reveals Cx43/hsa_circ_0077755/miR-182 as a biomarker axis for heightened risk of breast cancer initiation

doi: 10.1038/s41598-021-82057-y

Figure Lengend Snippet: Microarrays revealed 121 differentially expressed circRNAs in response to Cx43 silencing in Cx43-KO-S1 (pretumorigenic) cells versus S1 (nontumorigenic) breast epithelial cells in 3D. Triplicates of Cx43-KO-S1 and triplicates of S1 cells were plated on Matrigel™ for 11 days. Total RNA was extracted, digested with RNase R to remove linear RNAs and enrich circRNAs, reverse transcribed and hybridized to Arraystar Human circRNA Array V2 microarrays. ( a ) Box plot after quantile normalization showing the distributions of log2 ratios among the six samples. ( b ) Volcano plot depicting the differential circRNA expression, with the vertical green lines corresponding to 2.0-fold up and down, and the horizontal green line representing a p-value of 0.05. The red points in the plot represent the differentially expressed circRNAs with statistical significance. The circRNAs denoted in black font with arrows highlight the most up-regulated (right) and down-regulated (left) circRNAs, while the circRNAs denoted in red font with arrows highlight the three chosen and validated circRNAs in this study. ( c ) Bar graph showing the chromosomal distributions of the differentially expressed circRNAs. ( d ) Unsupervised hierarchical cluster analysis (heat map) of microarray data used to assess the significant expression of circRNAs when comparing Cx43-KO-S1 to S1 cells in 3D (the key range (6–10) represents the log2 value of the normalized intensity for each sample and not the fold change). “Red” indicates higher expression level, and “green” indicates lower expression level in Cx43-KO-S1 as compared to S1 cells. Each circRNA is represented by a single row of colored boxes and each sample is represented by a single column.

Article Snippet: To identify circRNA expression profile specific to the loss of Cx43, microarrays for circRNAs (Arraystar Human circRNA Array V2) were performed.

Techniques: Reverse Transcription, Expressing, Microarray

Journal: Scientific Reports

Article Title: A risk progression breast epithelial 3D culture model reveals Cx43/hsa_circ_0077755/miR-182 as a biomarker axis for heightened risk of breast cancer initiation

doi: 10.1038/s41598-021-82057-y

Figure Lengend Snippet: RT-qPCR validated nine significant differentially expressed circRNAs in the cultured epithelia. Four replicates of Cx43-KO-S1 and four replicates of S1 cells were plated in Matrigel for 11 days. Total RNA was extracted and RT-qPCR was performed in Cx43-KO-S1 versus S1 breast epithelial cells in 3D using 18S ribosomal RNA as an endogenous control for ( a ) the selected up-regulated circRNAs and ( b ) the selected down-regulated circRNAs and ( c ) the three Cx43 ( GJA1 ) derived circRNAs as per microarray results. Dot plot represents the mean fold change with the standard error of mean as error bars of each circRNA expression in the breast epithelial acini in 3D. The circRNAs highlighted in red font were confirmed to be significantly dysregulated in Cx43-KO-S1 as compared to S1 cells in 3D. *denotes p < 0.05 and **denotes p < 0.01 and *** denotes p < 0.001 for Cx43-KO-S1 versus S1 cells using one-tailed unpaired T-test.

Article Snippet: To identify circRNA expression profile specific to the loss of Cx43, microarrays for circRNAs (Arraystar Human circRNA Array V2) were performed.

Techniques: Quantitative RT-PCR, Cell Culture, Control, Derivative Assay, Microarray, Expressing, One-tailed Test

Journal: Scientific Reports

Article Title: A risk progression breast epithelial 3D culture model reveals Cx43/hsa_circ_0077755/miR-182 as a biomarker axis for heightened risk of breast cancer initiation

doi: 10.1038/s41598-021-82057-y

Figure Lengend Snippet: Sequencing revealed 29 significantly up-regulated and 36 significantly down-regulated mature miRNAs in Cx43-KO-S1 cells as compared to S1 cells in response to Cx43 silencing in cultured epithelia. Triplicates of Cx43-KO-S1 and triplicates of S1 cells were plated on Matrigel™ for 11 days. Total RNA was extracted, reverse transcribed and hybridized for sequencing using Illumina’s NovaSeq6000. ( a ) A heat map of unsupervised hierarchical clustering analysis shows for simplicity only miRNAs that were significantly detected from miRNA sequencing data (Fold Change > 2) and are in common with some of the five top MREs for each of the 121 significant differentially expressed circRNAs as predicted by Arraystar's miRNA target prediction software. Red depicts up-regulated miRNAs and blue depicts down-regulated ones in pretumorigenic Cx43-KO-S1 cells compared to nontumorigenic S1 counterparts. Samples were clustered using hierarchical clustering and miRNAs were similarly clustered using hierarchical clustering and are annotated with the direction (up or down-regulation) of the associated circRNAs in Cx43-KO-S1 samples versus S1 samples. Bright blue boxes annotate miRNAs that are predicted to bind to up-regulated circRNAs, whereas pink boxes annotate those associated with circRNAs that are down-regulated in Cx43-KO-S1 as compared to S1 acini. ( b ) A table showing the regulation pattern of miRNAs from miRNA sequencing of the 3D culture model that are in common with predicted MREs of only the 18 chosen circRNAs (from Tables , ), Fold change ≥ 1. The miRNAs in red font represent common significant miRNAs from miRNA sequencing results of 3D culture model and MREs that can be sponged by the nine validated circRNAs through RT-qPCR. Thus, these were selected for investigation in the potential post-transcriptional signature axes in the scope of this paper.

Article Snippet: To identify circRNA expression profile specific to the loss of Cx43, microarrays for circRNAs (Arraystar Human circRNA Array V2) were performed.

Techniques: Sequencing, Cell Culture, Reverse Transcription, Software, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: A risk progression breast epithelial 3D culture model reveals Cx43/hsa_circ_0077755/miR-182 as a biomarker axis for heightened risk of breast cancer initiation

doi: 10.1038/s41598-021-82057-y

Figure Lengend Snippet: Selection of one validated mRNA-circRNA-miRNA breast cancer initiation risk-assessment axis. ( a ) Comparative flow chart representing the dysregulation patterns of the validated circRNAs and that of their target miRNAs, based on (i) miRNA sequencing in Cx43-KO-S1 cells compared to S1 cells (shown in Fig. ) and (ii) tumor-associated miRNAs from microarrays of early-stage Lebanese breast cancer patient cohort as reported in Nassar et al. (shown in Table ). Only Cx43/has_circ_0077755/miR-182 axis exhibits the expected inverse dysregulation pattern between circRNA and their target miRNAs in both cells and patients (when circRNA is down-regulated, its MRE should be up-regulated, and vice versa). ( b ) RT-qPCR further confirmed the upregulation of miR-182 in four samples of Cx43-KO-S1 cells as compared to S1 counterparts using RNU6B as an endogenous control. * denotes a p.value < 0.05 for Cx43-KO-S1 versus S1 cells using one-tailed unpaired T-test. ( c ) Using METABRIC breast cancer miRNA dataset in the Kaplan–Meier Plotter , the survival analysis for miR-182 in 460 patients with grade II breast tumors was plotted. miR-182 seems to associate with poor prognosis when up-regulated in grade II breast tumors. ( d ) Using all breast cancer mRNA datasets in the Kaplan–Meier Plotter , , the survival analysis for Cx43 in 901 patients with grade II breast tumors was plotted. Cx43 seems to associate with poor prognosis when down-regulated . The same was performed for Grade III breast tumors and presented in (Supplementary Fig. a,b), where down-regulation of miR-182 and up-regulation of Cx43 seem to associate with poor prognosis in Grade III breast tumors.

Article Snippet: To identify circRNA expression profile specific to the loss of Cx43, microarrays for circRNAs (Arraystar Human circRNA Array V2) were performed.

Techniques: Selection, Sequencing, Quantitative RT-PCR, Control, One-tailed Test

Journal: Scientific Reports

Article Title: A risk progression breast epithelial 3D culture model reveals Cx43/hsa_circ_0077755/miR-182 as a biomarker axis for heightened risk of breast cancer initiation

doi: 10.1038/s41598-021-82057-y

Figure Lengend Snippet: Gene co-expression networks shows the involvement of the validated Cx43/has_circ_0077755/miR-182 axis in cancer-related pathways and in breast cancer. CircRNA-miRNA-mRNA gene co-expression network for hsa_circ_0077755 was predicted by TargetScan within IPA and Cytoscape was used to draw circRNA-miRNA-mRNA interaction networks. CircRNA is colored in green, miRNAs in pink and mRNAs reported in cancer in yellow and in breast cancer in purple. miR-182 exhibited the largest interaction network with mRNAs involved in cancer-related pathways in the axis.

Article Snippet: To identify circRNA expression profile specific to the loss of Cx43, microarrays for circRNAs (Arraystar Human circRNA Array V2) were performed.

Techniques: Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: The circular RNA Ataxia Telangiectasia Mutated regulates oxidative stress in smooth muscle cells in expanding abdominal aortic aneurysms

doi: 10.1016/j.omtn.2023.08.017

Figure Lengend Snippet: Circular RNAs are deregulated in human abdominal aortic aneurysm (A) Volcano plot depicting downregulated (51, blue) and upregulated (40, red). Circular RNAs (circRNAs) in human elective human abdominal aortic aneurysm (eAAA, n = 11) vs . control (CTRL, n = 6) aorta specimens, as resulted by array experiments. Log2 fold change and -log10 p value are plotted on the x and y axes, respectively. IDs of circRNAs meant for a first round of validation are highlighted. Statistics: unpaired t test; p values <0.05 are considered significant. (B) Pie chart illustrating the proportion of exonic (89.8%), intronic (5.7%), sense overlapping (3.4%), and antisense (1.1%) array-identified differentially expressed circRNAs. Absolute numbers are further indicated for each group. (C) Real-time quantitative PCR (qPCR) validation of hsa_circ_0005660 (c NFIX ), hsa_circ_0003641 (c ATM ), hsa_circ0042103 (c MYOCD ), hsa_circ003218 (c BMPR2 ), hsa_circ0004771 (c NRIP1 ), and hsa_circ0005615 (c NFATC3 ) differential expression in human eAAA (N = 8) and CTRL (N = 4) aortas. 2 –ddCT was calculated by normalizing on RPLPO . Data are represented as mean ± SEM. Statistics: unpaired t test; p values <0.05 are considered significant. NS, not significant; eAAA, elective AAA.

Article Snippet: The resulting labeled cDNA was then purified and 1 μg was fragmented, heated, and subsequently hybridized with an 8 × 15k commercially available array chip displaying 13,617 human circRNAs (Arraystar, no. AS-S-CR-H-V2.0) for 17 h at 65°C in an Agilent Hybridization Oven.

Techniques: Control, Biomarker Discovery, Real-time Polymerase Chain Reaction, Quantitative Proteomics