Journal: Respiratory Research

Article Title: CircATXN2 exacerbates pulmonary arterial hypertension by modulating the miR-138-5p/SMURF1 axis

doi: 10.1186/s12931-026-03647-w

Figure Lengend Snippet: CircATXN2 is upregulated in PAH models and hypoxic PASMCs A Right ventricular systolic pressure (RVSP) measured by invasive catheterization in control (Ctrl, normoxia) and Su-Hyp-induced PAH (PAH) mice ( n = 6). B , C Volcano plot (B) and Heatmap (C) of differentially expressed circRNAs from RNA-seq analysis of lung tissue from Ctrl and PAH mice (fold change (> 2 or < 0.5), P < 0.05). D qRT-PCR analysis of circATXN2 expression in primary mouse pulmonary artery endothelial cells (PAECs) versus pulmonary artery smooth muscle cells (PASMCs) ( n = 3). E qRT-PCR analysis of circATXN2 expression in PASMCs exposed to normoxia (Nor) or hypoxia (Hyp, 3% O2 for 24 h) ( n = 3) F RT-PCR validation of circATXN2 in PASMCs using divergent primers (amplifying circATXN2) and convergent primers (amplifying linear ATXN2 mRNA) with cDNA and genomic DNA (gDNA) templates. 18S was used as a positive control for both templates. G qRT-PCR analysis of circATXN2 and linear ATXN2 mRNA levels in PASMCs treated with RNase R or a mock control ( n = 3). H Schematic and Sanger sequencing validation of the back-splicing junction (BSJ) of circATXN2, confirming its origin from exons 18 and 19. I Fluorescence in situ hybridization (FISH) of circATXN2 (red) and 18S (green) in PASMCs, demonstrating primary cytoplasmic localization. Nuclei were counterstained with DAPI (blue). Scale bar: 50 µm. J RT-PCR analysis of the distribution of circATXN2 in diverse organs. K Conservation analysis of the circATXN2 sequence in different species. All data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 , ****P < 0.0001, ns , not significant

Article Snippet: After washing with PBS, the sections were incubated with a Cy3-conjugated anti-rabbit at RT for 1 h, followed by labeling the cell nuclei with DAPI (Servicebio).

Techniques: Control, RNA Sequencing, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Positive Control, Sequencing, Fluorescence, In Situ Hybridization

Journal: Respiratory Research

Article Title: CircATXN2 exacerbates pulmonary arterial hypertension by modulating the miR-138-5p/SMURF1 axis

doi: 10.1186/s12931-026-03647-w

Figure Lengend Snippet: CircATXN2 knockdown alleviates hypoxia-induced PASMC proliferation and migration. PASMCs were transfected with a negative control siRNA (si-NC) or an siRNA targeting circATXN2 (si-circATXN2) for 24 h. Cells were then exposed to normoxia (Nor) or hypoxia (Hyp, 3% O2) for an additional 24 h. A Viability measured by Cell Count Kit-8 (CCK-8) assays with transfected with si-circATXN2 ( n = 5) B , E Representative images ( B ) and quantification ( E ) of cell proliferation assessed by EdU incorporation. EdU-positive (proliferating) cells are green; nuclei are counterstained with DAPI (blue) ( n = 6). Scale bar: 100 µm. C , F Representative images ( C ) and quantification ( F ) of cell migration assessed by a wound healing assay. Images were captured at 0 h and 24 h post-scratch ( n = 5). Scale bar: 100 µm. D , G Representative images ( D ) and quantification ( G ) of cell migration assessed by a Transwell assay. Migrated cells were stained with crystal violet ( n = 5). Scale bar: 50 µm. All data are presented as the mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. * P < 0.05, **P < 0.01, ***P < 0.001, ns , not significant

Article Snippet: After washing with PBS, the sections were incubated with a Cy3-conjugated anti-rabbit at RT for 1 h, followed by labeling the cell nuclei with DAPI (Servicebio).

Techniques: Knockdown, Migration, Transfection, Negative Control, Cell Characterization, CCK-8 Assay, Wound Healing Assay, Transwell Assay, Staining

Journal: Respiratory Research

Article Title: CircATXN2 exacerbates pulmonary arterial hypertension by modulating the miR-138-5p/SMURF1 axis

doi: 10.1186/s12931-026-03647-w

Figure Lengend Snippet: circATXN2 acting as a miR-138-5p sponge. A RNA Immunoprecipitation (RIP) for AGO2. qRT-PCR shows significant enrichment of circATXN2 in AGO2 pulldowns compared to IgG controls in PASMCs ( n = 3). B Venn diagram of predicted miRNA targets for circATXN2 from miRanda, TargetScan, and RNAhybrid, identifying miR-138-5p as a top candidate. C RNA pulldown assay using biotinylated miR-138-5p or other candidate miRNAs identified in (B). qRT-PCR analysis shows specific and significant enrichment of circATXN2 only with the miR-138-5p probe ( n = 3). D Schematic of circATXN2 (318 nt) showing the 6 predicted binding sites for miR-138-5p. E Co-localization of circATXN2 (red) and miR-138-5p (green) in PASMCs via FISH assays, demonstrating co-localization in the cytoplasm. Nuclei are counterstained with DAPI (blue). Scale bar: 50 µm. F , G qRT-PCR analysis of miR-138-5p levels in PASMCs following (F) knockdown of circATXN2 (si-circATXN2) or (G) overexpression of circATXN2 (oe-circATXN2), demonstrating reciprocal regulation ( n = 3). H-M Functional rescue experiments. PASMCs were co-transfected with a miR-138-5p mimic or negative control (miR-NC), along with an overexpression vector for circATXN2 (oe-circATXN2) or its control (oe-NC). CCK-8 viability assay ( n = 5) (H). Representative images (I) and quantification (K) of EdU proliferation assay ( n = 3). Scale bar: 100 µm. Representative images (J) and quantification (M) of Transwell migration assay ( n = 3). Scale bar: 50 µm. Quantification of wound healing migration assay ( n = 3) (L). The miR-138-5p mimic reversed the pro-proliferative and pro-migratory effects of circATXN2 overexpression. All data are presented as the mean ± SEM. Statistical significance was determined using a two-tailed Student's t-test (for A, C, F, G) or one-way ANOVA followed by Tukey's post-hoc test (for H, K, L, M). * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns , not significant

Article Snippet: After washing with PBS, the sections were incubated with a Cy3-conjugated anti-rabbit at RT for 1 h, followed by labeling the cell nuclei with DAPI (Servicebio).

Techniques: RNA Immunoprecipitation, Quantitative RT-PCR, Binding Assay, Knockdown, Over Expression, Functional Assay, Transfection, Negative Control, Plasmid Preparation, Control, CCK-8 Assay, Viability Assay, Proliferation Assay, Transwell Migration Assay, Migration, Two Tailed Test

Journal: Respiratory Research

Article Title: CircATXN2 exacerbates pulmonary arterial hypertension by modulating the miR-138-5p/SMURF1 axis

doi: 10.1186/s12931-026-03647-w

Figure Lengend Snippet: Reducing circATXN2 in SMCs attenuated the severity of PAH via miR-138-5p/SMURF1 axis. A Immunofluorescence staining of pulmonary arteries confirming SMC-specific AAV9 transduction. GFP (AAV9, green) co-localizes with the SMC marker α-SMA (red). Nuclei were counterstained with DAPI (blue). Scale bar: 100 µm. B qRT-PCR analysis of circATXN2 expression in lung tissue from mice injected with AAV9-si-NC or AAV9-si-circATXN2, confirming successful knockdown ( n = 6) . C Quantification of Right Ventricular Systolic Pressure (RVSP) in the four treatment groups ( n = 6). D , E Echocardiographic measurements of AT/ET ratio (D) and PAVTI (E) in the four treatment groups ( n = 6). F , G Quantification (F) and Hematoxylin and Eosin (H&E) staining (G) of pulmonary arterioles, showing reduced medial wall thickening in the AAV9-si-circATXN2-treated hypoxia group ( n = 6). Scale bar: 50 µm. H Western blot analysis of SMURF1 protein expression in lung lysates from the four treatment groups. β-actin was used as a loading control ( n = 6). I To validate the in vivo role of circATXN2, SMC-specific knockdown was achieved using AAV9-si-circATXN2 in 6-week-old Tagln-Cre mice. As shown in the schematic, mice were injected via the tail vein. After a two-week infection period, mice were exposed to normoxia (21% FiO 2 ) or chronic hypoxia (10% FiO 2 ) and weekly subcutaneous semaxanib (SU5416, 20 mg/kg/week) injections for 4 weeks to induce PAH. All data are presented as the mean ± SEM. Statistical significance was determined using a two-tailed Student's t-test (for B) or two-way ANOVA followed by Tukey's post-hoc test (for C-F). * P < 0.05, **P < 0.01, ns , not significant

Article Snippet: After washing with PBS, the sections were incubated with a Cy3-conjugated anti-rabbit at RT for 1 h, followed by labeling the cell nuclei with DAPI (Servicebio).

Techniques: Immunofluorescence, Staining, Transduction, Marker, Quantitative RT-PCR, Expressing, Injection, Knockdown, Western Blot, Control, In Vivo, Infection, Two Tailed Test