Journal: JACC: Basic to Translational Science

Article Title: Human Left Ventricle circRNA-miRNA-mRNA Network Analyses Reveal a Novel Proangiogenic Role for circNPHP1 Under Ischemic Conditions

doi: 10.1016/j.jacbts.2025.101468

Figure Lengend Snippet: CircNPHP1 Expression in the Human LV and Cultured ECs Cardiac CircNPHP1 levels are higher in patients with IHD with/without associated T2DM and in cultured endothelial cells (human cardiac microvascular endothelial cells and human umbilical vein endothelial cells) exposed to disease-mimicking conditions, such as hypoxia and hypoxia, combined with increased D-glucose level (High glu). (A) Preliminary screening of circRNAs (identified from predicted networks) in endothelial cells: human cardiac microvascular endothelial cells were cultured in normal conditions, hypoxia (1% Oxygen), and hypoxia-high glucose (High Glu) (25 mmol/L D-glucose) conditions, respectively. After 48 hours, cells were harvested for quantitative real-time polymerase chain reaction (qRT-PCR) for the analysis and validation of indicated circRNAs. RNA expression is relative to normoxia. 18S is used as housekeeping gene; n = 3. Data are expressed as mean ± SEM and were assessed by 1-way analysis of variance with Dunnett's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ns = not significant. CircSTX17 was also tested but the levels were undetectable; hence, it is not included in the plot. CircNPHP1 emerges as an interesting candidate for further exploration and validation. (B) Comparison of expression levels of circNPHP1 and its linear counterpart in different patient groups—RNAseq (LV biopsies, cohort 1). Log-transformed normalized (FPM) counts of circNPHP1 and linear NPHP1 are plotted as a boxplot showing the median and IQR. Comparison between patient groups and calculation of P values was done using the R package Limma. CircNPHP1 is significantly up-regulated in IHD+T2DM compared with IHD and non-IHD ( P < 0.05). Linear NPHP1 is expressed at very low levels and does not change between the different patient groups (IHD [n = 12], IHD+T2DM [n = 11], non-IHD [n = 12]). (C) qRT-PCR of circNPHP1 (left) and linear NPHP1 (right) of LV biopsies (cohort 1) from non-IHD (n = 12), IHD (n = 12), and IHD+T2DM (n = 11), and (D) of LV biopsies (cohort 2) from non-IHD (n = 10), IHD (n = 10), IHD+T2DM (n = 10) of patients. RNA expression is relative to non-IHD; 18S is used as housekeeping gene. Data are expressed as mean ± SEM and were assessed by Kruskal-Wallis test followed by Dunn's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ns = not significant. Abbreviations as in .

Article Snippet: For the analysis of circRNAs, cDNA was synthesized using the PrimeScript qRT-PCR kit (Takara), according to the manufacturer’s instructions. qRT-PCR was performed using TB Green Premix Ex Taq Kit (Takara) on QuantStudio 6 Flex Real-Time PCR System (Life Technologies).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Biomarker Discovery, RNA Expression, Comparison, Transformation Assay

Journal: JACC: Basic to Translational Science

Article Title: Human Left Ventricle circRNA-miRNA-mRNA Network Analyses Reveal a Novel Proangiogenic Role for circNPHP1 Under Ischemic Conditions

doi: 10.1016/j.jacbts.2025.101468

Figure Lengend Snippet: Identification and Validation of the CircNPHP1 Proangiogenic Interactome in Endothelial Cells CircNPHP1 is physically associated with the antiangiogenic miR-221-3p to putatively command a proangiogenic subnetwork. (A) CircNPHP1 subnetwork extracted from T2DM network (left). CircNPHP1 is connected to many miRNAs and mRNAs (circled in black) that mapped to EC-relevant pathways (VEGF signaling and angiogenesis; right, top). In particular, miR-221-3p bound exclusively to circNPHP1 via its back-splice junction sequence (right, bottom). The arc consisting of circNPHP1, its sponged miRNA partners (miR-221-3p, miR-222-3p, miR-299-3p, miR-141-3p, and miR-139-5p) and downstream targets (VEGFA, BCL2, BCL2L11) were selected for further validation. (B) CircNPHP1 ASO (anti-sense oligonucleotide) pulldown assay: HUVEC cell extracts were incubated with biotinylated ASO targeting the back-splice junction of circNPHP1 (probes 1 and 2) or control probe (nontargeting sequence) (see for the details). Subsequently, the pulldown of endogenous circNPHP1 was carried out using streptavidin beads (schematic representation; see details in Methods section). The precipitated RNA was subjected to quantitative real-time polymerase chain reaction (qRT-PCR) for the analysis of circNPHP1 (left) and miR-221-3p (right). Fold enrichment was calculated against the control probe pulldown. 18S and U6 were used as reference genes to normalize for circNPHP1 and miR-221-3p respectively (n = 3). (C) HUVECs were transfected with 30 nmol/L of circNPHP1 siRNA (siRNA1) or control siRNA. 48 hours post-transfection, cells were harvested and incubated with biotinylated ASO control probe, probe 1, and probe 2, respectively, for the pulldown of circNPHP1. Subsequently, the precipitated RNA was subjected to qRT-PCR for the analysis of circNPHP1, linear NPHP1, and miR-221-3p. Fold enrichment was calculated relative to the control probe pulldown. 18S and U6 were used as reference genes to normalize for circNPHP1, linear NPHP1, and miR-221-3p, respectively (n = 3). Data (B and C) are expressed as mean ± SEM and were assessed by 1-way analysis of variance with Dunnett's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ns = not significant. (D) miRNA pulldown assay: HUVECs were transfected with 50 nmol/L of biotinylated miRNAs (miR-221-3p, miR-222-3p, miR-139-5p, respectively) or control biotinylated miRNA (scrambled nontargeting sequence). 48 hours post-transfection, cells were harvested and incubated with streptavidin beads for pulldown of the miRNAs (schematic representation; see details in Methods section). Subsequently, the precipitated RNA was subjected to qRT-PCR for the analysis of circNPHP1 (left) and the miRNAs (right). Fold enrichment (n = 3) was calculated in the pulldown samples as follows: miRNA pulldown/ control pulldown (X); miRNA input/ control input (Y), fold enrichment = X/Y. 10% of the cell extract was used as input for individual samples. Data are expressed as mean ± SEM and were assessed by unpaired Student's t -test between control and miRNA pulldown group. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ns = not significant. Abbreviations as in .

Article Snippet: For the analysis of circRNAs, cDNA was synthesized using the PrimeScript qRT-PCR kit (Takara), according to the manufacturer’s instructions. qRT-PCR was performed using TB Green Premix Ex Taq Kit (Takara) on QuantStudio 6 Flex Real-Time PCR System (Life Technologies).

Techniques: Biomarker Discovery, Sequencing, Incubation, Control, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection