Journal: Experimental & Molecular Medicine

Article Title: Circular RNA circ-4099 is induced by TNF-α and regulates ECM synthesis by blocking miR-616-5p inhibition of Sox9 in intervertebral disc degeneration

doi: 10.1038/s12276-018-0056-7

Figure Lengend Snippet: Top 10 differentially expressed circRNAs (degenerated vs. non-degenerated)

Article Snippet: This array is the first commercially available circRNA microarray (Arraystar, Inc., USA) designed for the global profiling of human circRNAs.

Techniques:

Journal: Experimental & Molecular Medicine

Article Title: Circular RNA circ-4099 is induced by TNF-α and regulates ECM synthesis by blocking miR-616-5p inhibition of Sox9 in intervertebral disc degeneration

doi: 10.1038/s12276-018-0056-7

Figure Lengend Snippet: a Scatter plot of circRNA expression. The circRNAs above the top green line and below the bottom green line indicated more than a twofold change between the degenerated and normal IVD NP tissues. b Volcano Plot of the differentially expressed circRNAs. The red points in the plot represent differentially expressed circRNAs with statistical significance. c Hierarchical clustering shows the distinguishable circRNA expression profile between the two groups and homogeneity within the groups. d The sequence of the amplified Circ-4099 product was completely consistent with CircBase. e Co-localization between circ-4099 (red) and miR-616-5p (green) was observed by RNA in situ hybridization in hNP cells after co-transfection with circ-4099-expressing and miR-616-5p-expressing vectors. The nuclei were stained with DAPI (blue). Scale bar = 10 µm. f The expression levels of circ-4099 are significantly higher in degenerated NP tissues than in non-degenerated NP tissues. g – i The expression levels of circ-68610/2069/92328 are higher in degenerated NP tissues than in non-degenerated NP tissues. j qRT-PCR analysis shows that TNF-α upregulates the expression of circ-4099 in a dose-dependent and time-dependent manner. For time-dependent studies, 50 ng/ml TNF-α was used. k hNP cells were transfected with the circ-4099-Luc promoter and the induction in luciferase activity was measured following TNF-α treatment ( P < 0.001)

Article Snippet: This array is the first commercially available circRNA microarray (Arraystar, Inc., USA) designed for the global profiling of human circRNAs.

Techniques: Expressing, Sequencing, Amplification, RNA In Situ Hybridization, Cotransfection, Staining, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay

Journal: Experimental & Molecular Medicine

Article Title: Circular RNA circ-4099 is induced by TNF-α and regulates ECM synthesis by blocking miR-616-5p inhibition of Sox9 in intervertebral disc degeneration

doi: 10.1038/s12276-018-0056-7

Figure Lengend Snippet: a Plasmid map of the Circ-4099 over-expression plasmid. b The qPCR products were used to confirm the Circ-4099 sequence. c , d hNP cells were transfected with Circ-4099, NC (PLCDH-ciR) or negative control, and total RNA was collected. qPCR was used to detect the expression of Circ-4099 and its linear RNA (DENND5A) (linear + circRNA). The results showed that the Circ-4099 over-expression plasmid can extremely enhance Circ-4099 expression. These data confirm the validity and efficiency of the Circ-4099 over-expression plasmid. e , f qRT-PCR showed that Circ-4099 over-expression led to increased Collagen II and Aggrecan mRNA expression, which was suppressed by treatment with miR-616-5p mimics, while MMP3 and ADAMTS5 showed few significant differences in regulation. g , h Western blotting also showed that he Collagen II and Aggrecan protein expression levels were significantly increased by circ-4099 over-expression treatment. We next tested the effects of Circ-4099 on expression of inflammatory cytokines in hNP. i , j The IL-1β, TNF-α, and PGE2 mRNA levels were significantly suppressed in hNP cells transduced with Circ-4099 over-expression plasmid, which was inhibited by treatment with miR-616-5p mimics. k , l IL-1β, TNF-α, IL-6, and PGE2 cytokine release into medium was detected by ELISA, which showed similar results

Article Snippet: This array is the first commercially available circRNA microarray (Arraystar, Inc., USA) designed for the global profiling of human circRNAs.

Techniques: Plasmid Preparation, Over Expression, Sequencing, Transfection, Negative Control, Expressing, Quantitative RT-PCR, Western Blot, Transduction, Enzyme-linked Immunosorbent Assay

Journal: Non-coding RNA Research

Article Title: Circular RNAs in intracranial aneurysms: Emerging roles in pathogenesis, diagnosis and therapeutic intervention

doi: 10.1016/j.ncrna.2023.11.012

Figure Lengend Snippet: Schematic illustration of the effect of circular RNAs (circRNAs) on intracranial aneurysm (IA) rupture. Current evidence strongly suggests a central role for endothelial dysfunction in the initiation and progression of IA. Post-subarachnoid hemorrhage (SAH), several early pathophysiological events can be commonly observed in blood-brain barrier (BBB) components, such as the endothelium (endothelial dysfunction). In results, post- SAH injuries can disrupt the integrity and function of the BBB . Both negative (red cross) and positive (green cross) regulation of circRNAs have been observed in this pathological cascade. The role of circRNAs is based on components: 1) strong role in endothelial cells (ECs) homeostasis; 2) regulation of barrier function and vascular tone; 3) associated with SAH and its complications; 4) correlates with clinical outcomes (Glasgow Coma Scale, the volume of SAH, modified Fisher scale, Hunt-Hess levels, and surgical type; 5) regulators of transcription/translation, sequesters of microRNA (miRNA)/RNA-binding proteins (RBPs), and biomarkers of IA.

Article Snippet: Leveraging the power of the Arraystar human circRNAs microarray, differentially expressed circRNAs between patients with UIAs and RIAs were meticulously analyzed.

Techniques: Modification, RNA Binding Assay

Journal: Non-coding RNA Research

Article Title: Circular RNAs in intracranial aneurysms: Emerging roles in pathogenesis, diagnosis and therapeutic intervention

doi: 10.1016/j.ncrna.2023.11.012

Figure Lengend Snippet: Schematic illustration of circular RNAs (circRNAs) regulation mechanisms underlying vascular smooth muscle cells (VSMCs) phenotypic modulation, oxidative stress, and cell death in intracranial aneurysms (IAs). As can be seen from the figure, circRNAs play a role both in the development and progression of IA and in the inhibition of IA through the control of VSMC. However, some of them exhibit a double effect as circ_FOXO3 and circ_0020397.

Article Snippet: Leveraging the power of the Arraystar human circRNAs microarray, differentially expressed circRNAs between patients with UIAs and RIAs were meticulously analyzed.

Techniques: Inhibition

Journal: Non-coding RNA Research

Article Title: Circular RNAs in intracranial aneurysms: Emerging roles in pathogenesis, diagnosis and therapeutic intervention

doi: 10.1016/j.ncrna.2023.11.012

Figure Lengend Snippet: Benefits of using cell free circular RNAs (circRNAs) as biomarkers.

Article Snippet: Leveraging the power of the Arraystar human circRNAs microarray, differentially expressed circRNAs between patients with UIAs and RIAs were meticulously analyzed.

Techniques:

Journal: Non-coding RNA Research

Article Title: Circular RNAs in intracranial aneurysms: Emerging roles in pathogenesis, diagnosis and therapeutic intervention

doi: 10.1016/j.ncrna.2023.11.012

Figure Lengend Snippet: The studied cell free circular RNAs (circRNAs) are presented as non-invasive biomarkers in intracranial aneurysms (IAs).

Article Snippet: Leveraging the power of the Arraystar human circRNAs microarray, differentially expressed circRNAs between patients with UIAs and RIAs were meticulously analyzed.

Techniques:

Journal: Non-coding RNA Research

Article Title: Circular RNAs in intracranial aneurysms: Emerging roles in pathogenesis, diagnosis and therapeutic intervention

doi: 10.1016/j.ncrna.2023.11.012

Figure Lengend Snippet: Summary information on the role of circular RNAs (circRNAs) in the formation and development of intracranial aneurysms (IAs).

Article Snippet: Leveraging the power of the Arraystar human circRNAs microarray, differentially expressed circRNAs between patients with UIAs and RIAs were meticulously analyzed.

Techniques: Migration, Transformation Assay

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: Aging (Albany NY)

Article Title: Plasmatic exosome-derived circRNAs panel act as fingerprint for glioblastoma

doi: 10.18632/aging.203368

Figure Lengend Snippet: The hsa_circ_0055202, hsa_circ_0074920 and hsa_circ_0043722 promoted cell proliferation of GBM cell lines. ( A ) Relative expression of hsa_circ_0055202, hsa_circ_0074920 and hsa_circ_0043722 in GBM cell lines. ( B ) Relative expression of hsa_circ_0055202, hsa_circ_0074920 and hsa_circ_0043722 in cells treated with shRNA. ( C ) CCK8 assay in cells treated with circRNA shRNA. * indicated P < 0.05, ** indicated P < 0.01.

Article Snippet: The microarray-based circRNA expression profiling was conducted by Capitalbio (Beijing, China) using plasma RNA samples.

Techniques: Expressing, shRNA, CCK-8 Assay