CircIdh2 Attenuates Endothelial‐to‐Mesenchymal Transition and Myocardial Fibrosis and Exhibits Translational Potential

Figures

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  Figure 1. CircIdh2 expression was downregulated in TGF‐β1–induced rEPCs. (A) rEPCs were treated with TGF‐β1 (5 ng/mL) for 7 days. qRT‐PCR analysis of circSorbs1, circIdh2, and circSntb2. (B) Agarose gel electrophoresis was used to detect the PCR products of circIdh2, Idh2, and GAPDH using cDNA and gDNA templates. (C) Sanger sequencing was used to detect the back‐splice junction of circIdh2. (D) Rat circIdh2 was formed by back‐splicing of exons 1, 2, 3, and 11 from Idh2 pre‐mRNA; the human IDH2 locus is shown at chr15(q26.1). (E) FISH showed that circIdh2 was mainly localized in the cytoplasm. Scale bars, 25 μm. Data are mean ± SD; n = 3 independent biological experiments. Two‐way ANOVA followed by Šídák′s multiple‐comparisons test was used to compare NC and TGF‐β1 within each circRNA; ∗∗∗∗p < 0.0001.

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  Figure 2. circIdh2 attenuated TGF‐β1–induced EndMT changes in rEPCs. rEPCs were treated with or without TGF‐β1 (5 ng/mL) for 7 days, and then transfected with PCDH‐NC or PCDH‐circIdh2. (A) qRT‐PCR analysis of circIdh2 expression after transfection for 24 h. (B) Western blot analysis of EndMT‐related proteins (VE‐cadherin, vimentin, and N‐cadherin) after transfection for 48 h. (C) Transwell assay was performed to assess cell migration after transfection for 24 h. (D) Representative scratch wound healing images at 0, 24, and 48 h. Scale bars, 200 μm. (E) Quantification of relative wound width (% of PCDH‐NC at 0 h). Data are mean ± SD; n = 3 independent biological experiments; one‐way ANOVA; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

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  Figure 3. Knockdown of circIdh2 promoted EndMT changes in rEPCs. rEPCs were transfected with si‐NC or siRNAs targeting circIdh2. (A) qRT‐PCR analysis of circIdh2 expression after transfection for 24 h. (B) Western blot analysis of EndMT‐related proteins (VE‐cadherin, vimentin, and N‐cadherin) after transfection for 48 h. (C) Cell migration was assessed using a Transwell assay after transfection for 24 h. (D) Representative scratch wound healing images were obtained at 0, 24, and 48 h. Scale bars, 200 μm. (E) Quantification of relative wound width (% of si‐NC at 0 h). Data are mean ± SD; n = 3 independent biological experiments; Student′s t‐test was used for two‐group comparisons and one‐way ANOVA for multiple‐group comparisons; ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

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  Figure 4. circIdh2 showed translational potential and a FLAG‐tagged product was detected in an overexpression system. (A) Luciferase activity of the Idh2‐IRES element and its mutant form. (B) Co‐IP followed by western blotting was used to detect FLAG‐tagged protein expression, and (C) Coomassie Brilliant Blue staining showed the protein region selected for mass spectrometry. (D) Mass spectrometry results showing the peptide sequence corresponding to the putative circIdh2‐derived product and the representative MS/MS spectrum. Data are mean ± SD; n = 3 independent experiments; one‐way ANOVA; ∗∗p < 0.01 and ∗∗∗∗p < 0.0001.

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  Figure 5. The FLAG‐tagged circIdh2 construct affected EndMT‐related markers and cell migration in rEPCs. rEPCs were transfected with PCDH‐NC or PCDH‐flag‐circIdh2. (A) qRT‐PCR analysis of circIdh2 expression after transfection for 24 h. (B) Western blot analysis of FLAG expression after transfection for 48 h. (C) Western blot analysis of EndMT‐related proteins (VE‐cadherin, vimentin, and N‐cadherin) after transfection for 48 h. (D) Cell migration was examined by Transwell assay after transfection for 24 h. (E) Representative images from the scratch wound healing assay at 0, 24, and 48 h. Scale bar, 200 μm. (F) Quantification of relative wound width (% of PCDH‐NC at 0 h). Data are mean ± SD; n = 3 independent biological experiments; unpaired Student′s t‐test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

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  Figure 6. The circIdh2 mutant did not reproduce the effects of wild‐type circIdh2 on EndMT‐related markers and cell migration. rEPCs were transfected with PCDH‐NC, PCDH‐circIdh2, or PCDH‐circIdh2‐mut. (A) Western blot analysis of EndMT‐related proteins (VE‐cadherin, vimentin, and N‐cadherin) after transfection for 48 h. (B) Cell migration was assessed using a Transwell assay after transfection for 24 h. (C) Representative scratch wound healing images at 0, 24, and 48 h, with quantification of relative wound width (% of PCDH‐NC at 0 h). Scale bar, 200 μm. Data are mean ± SD; n = 3 independent biological experiments; one‐way ANOVA; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

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  Figure 7. AAV‐mediated delivery of circIdh2 or circIdh2‐aa attenuated myocardial fibrosis and EndMT‐related marker changes in vivo. (A) HE staining of myocardial infarction tissues from the sham, model, circIdh2‐AAV, and circIdh2‐aa‐AAV groups. (B) Masson staining of MI tissues. (C) IF detection and quantification of fibrotic markers (Collagen I and TGF‐β) in myocardial tissues. Scale bars, 100 μm. (D) Western blot analysis of EndMT‐related proteins (VE‐cadherin, vimentin, and N‐cadherin). Data are mean ± SD; n = 3 rats per group; one‐way ANOVA; ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.