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

Figures

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  Fig. 1. 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…

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  Fig. 2. 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

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  Fig. 3. 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…

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  Fig. 4. miR-138-5p directly targets and suppresses SMURF1 A Venn diagram of predicted mRNA targets for miR-138-5p from five algorithms, identifying SMURF1 as a candidate. B qRT-PCR analysis of top candidate mRNAs in PASMCs 48 h after transfection with a miR-138-5p mimic or negative control (miR-NC). SMURF1 is the only target significantly downregulated (n = 3). C, D Representative Western blot (C) and quantification (D) of SMURF1 protein levels in PASMCs transfected with miR-NC, miR-138-5p mimic, or miR-138-5p inhibitor. β-actin was used as a loading control (n = 4). E–G Representative Western blot (E) and quantification (F, G) of SMAD1 protein levels in PASMCs following knockdown (si-SMURF1) or overexpression (oe-SMURF1) of SMURF1, demonstrating that SMURF1 negatively regulates SMAD1 (n = 3). H Dual-luciferase reporter assay in 293 T cells. Cells were co-transfected with the miR-138-5p mimic and a pGL3 vector containing the wild-type SMURF1 3'-UTR (3'UTR-WT) or a vector with a mutated seed-site (3'UTR-MUT). The mimic significantly suppressed luciferase activity of the WT vector but not the MUT vector (n = 3). I-L Functional rescue experiments. PASMCs were co-transfected with a si-ATXN2…

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  Fig. 5. 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…

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  Fig. 6. Schematic diagram summarizing the findings. Under hypoxic conditions, circATXN2 expression is upregulated in PASMCs. circATXN2, which is formed by the back-splicing of ATXN2 exons, acts as a ceRNA to sponge miR-138-5p. This sequestration of miR-138-5p relieves its suppression of SMURF1 mRNA, leading to increased SMURF1 protein expression. Elevated SMURF1, in turn, promotes the degradation of R-SMADs, thereby impairing the anti-proliferative BMP signaling pathway. The disruption of this pathway results in enhanced PASMC proliferation and migration, which contributes to pulmonary vascular remodeling and the pathogenesis of PAH