The circular RNA circNlgnmediates doxorubicin-inducedcardiac remodeling and fibrosis

Figures

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  Figure 1. Doxorubicin-induced cardiac fibrosis and impaired heart function were exacerbated by circNlgn expression (A) Left, circNlgn transgenic mice, circNlgn (+), showed decreased left-ventricular ejection fraction (LVEF) relative to the litter-matched negative mice, circNlgn (−), after doxorubicin treatment. ∗∗p < 0.01 versus circNlgn (−) (n = 10). Right, circNlgn (+) mice showed decreased left-ventricular fractional shortening (LVFS) compared with circNlgn (−) after doxorubicin treatment. ∗∗p < 0.01 versus circNlgn (−) (n = 10). (B) circNlgn (+) mice displayed lower left-ventricular pressure (dp/dt) than circNlgn (−) mice after doxorubicin treatment. ∗∗p < 0.01 versus circNlgn (−) (n = 10). (C) circNlgn (+) mice displayed higher heart weight/tibia length (left) and heart weight/body weight (right) than circNlgn (−) mice after doxorubicin treatment. ∗∗p < 0.01 versus circNlgn (−) (n = 10). (D) Representative echocardiography photographs showing heart function of circNlgn (+) and circNlgn (−) mice with or without doxorubicin treatment. (E) Representative H&E staining of cross sections of heart tissues. circNlgn (+) mice displayed enlarged heart chamber. (F) circNlgn (+) mouse hearts…

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  Figure 2. Silencing circNlgn decreased the effects of doxorubicin on cardiac cell activity and prevented doxorubicin-induced expression of fibrosis-associated molecules (A) Left, real-time PCR showed that AC16 cells expressed increased circNlgn when cultured in basal medium and treated with doxorubicin for 1 h. ∗p < 0.05, ∗∗p < 0.01 versus doxorubicin 0 μM (n = 4). Right, real-time PCR showed that MCFs expressed increased levels of circNlgn when treated with doxorubicin. ∗p < 0.05, ∗∗p < 0.01 versus doxorubicin 0 μM (n = 4). (B) AC16 cells transfected with circNlgn or the vector were cultured in basal medium treated with doxorubicin for survival assay. circNlgn expression repressed cell survival. ∗∗p < 0.01 versus vector (n = 4). (C) Expression of circNlgn promoted cell apoptosis when treated with doxorubicin, which was dose dependent. ∗p < 0.05, ∗∗p < 0.01 versus vector (n = 4). (D) Silencing circNlgn enhanced AC16 cell survival. ∗p < 0.05, ∗∗p < 0.01 versus oligo (n = 4). (E) Silencing circNlgn repressed AC16 apoptosis. ∗∗p < 0.01 versus oligo (n = 4). (F) MCF cells transfected with circNlgn or the vector were cultured in basal medium for the indicated time points. Expression of circNlgn…

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  Figure 3. Nlgn173 binds and activates H2AX, producing γH2AX (A) Mass spectrophotometry showed proteins precipitated by antibody against Nlgn173. (B) AC16 cells transfected with circNlgn or the vector were lysed and subjected to immunoprecipitation with anti-Nlgn antibody followed by western blotting. Precipitation of Nlgn173 pulled down H2AX, and vice versa. (C) The interaction of Nlgn173 and H2AX promoted phosphorylation of H2AX (γH2AX). (D) AC16 cells transfected with circNlgn expressed higher levels of Nlgn173 and γH2AX, which were further enhanced by doxorubicin treatment. H2AX expression was not affected. (E) Precipitation of H2AX pulled down Nlgn173. H2AX phosphorylation (γH2AX) was higher in the sample where Nlgn173 interacted with H2AX. (F) Precipitation of Nlgn173 pulled down H2AX. The interaction of Nlgn173 and H2AX promoted phosphorylation of H2AX (γH2AX). (G) Western blot showed that circNlgn (+) mouse tissues expressed high levels of Nlgn173 and γH2AX, which were further enhanced by doxorubicin treatment. (H) Quantitation of IHC staining of heart tissues showed increased expression of Nlgn173 in the circNlgn transgenic heart, which could be further promoted by doxorubicin…

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  Figure 4. Upregulation of Gadd45b, Sema4C, and RAD50 in circNlgn transgenic heart (A) Volcano analysis of 24,283 mRNAs detected by RNA sequencing. Approximately 3% of the RNAs were differentially expressed. (B) Heatmap analysis showing the differentially expressed RNAs. (C) From the 307 differentially expressed mRNAs, the most up- and downregulated were selected. (D) PCR showed that PCMs from the hearts of the circNlgn-transgenic mice expressed significantly higher levels of circNlgn, Gadd45b, Sema4C, RAD50, IL-1b, IL-1rap, IL-2Rb, IL-6, EGR1, and EGR3. ∗p < 0.05, ∗∗p < 0.01 versus wild type (n = 4). (E) PCR showed that circNlgn (+) mouse heart tissues expressed high levels of circNlgn, Gadd45b, Sema4C, RAD50, IL-1b, IL-2Rb, IL-6, EGR1, and EGR3, which were further enhanced by delivery of doxorubicin. ∗∗p < 0.01 (n = 4).

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  Figure 5. Dissecting out the downstream signal molecules of γH2AX (A) PCR showed that PCMs transfected with circNlgn siRNA repressed expression of circNlgn, Gadd45b, Sema4C, RAD50, IL-1b, IL-2Rb, IL-6, EGR1, and EGR3. IL-1rap did not show downregulation upon circNlgn silencing. ∗p < 0.05, ∗∗p < 0.01 versus oligo (n = 4). (B) Silencing EGR1 (left) and EGR3 (right) did not affect expression of any other molecules. ∗∗p < 0.01 versus oligo (n = 4). (C and D) Silencing IL-1b decreased expression of EGR3 (C), while silencing IL-2Rb decreased expression of EGR1 (D). ∗∗p < 0.01 versus oligo (n = 4). (E) Silencing IL-6 decreased expression of EGR1 and EGR3. ∗p < 0.05, ∗∗p < 0.01 versus oligo (n = 4). (F and G) Silencing Gadd45b (F) or Sema4C (G) repressed expression of all these molecules (IL-1b, IL-2Rb, IL-6, EGR1, and EGR3). ∗p < 0.05, ∗∗p < 0.01 versus oligo (n = 4). (H) Silencing RAD50 did not affect expression of any molecules tested. ∗∗p < 0.01 versus oligo (n = 4).

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  Figure 6. Effects of the signaling molecules in mediating cellular functions of circNlgn (A) Western blot showed that circNlgn (+) heart tissues expressed high levels of Nlgn173, γH2AX, pJNK, and p38, which were further promoted by delivery of doxorubicin. (B) PCMs were transfected with circNlgn siRNAs and cultured with basal medium with 0.2 μM doxorubicin for 36 h. Silencing circNlgn suppressed Nlgn173, γH2AX, pJNK, and p38 expression. (C) Control vector- and circNlgn-transfected cells were treated with or without 0.25 μM doxorubicin for 24 h, lysed, and subjected to western blotting. circNlgn transfection increased the levels of Nlgn173, γH2AX, pJNK, and p38, which were further promoted by doxorubicin treatment. (D) Signaling pathway of circNlgn for mediating doxorubicin effect on cardiac remodeling, fibrosis, and cardiomyopathy. (E) Western blot showed that circNlgn (+) PCMs expressed high levels of γH2AX, pJNK, and p38. Silencing Gadd45b prevented circNlgn-enhanced γH2AX, pJNK, and p38. Silencing Sema4C prevented circNlgn-enhanced γH2AX and p38, but not pJNK.

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  Figure 7. Activation of H2AX by p38 and pJNK (A) PCMs were transfected with siRNAs and cultured in basal medium with 2 μM doxorubicin for 24 h (left) or serum free for 5 days (right). Silencing circNlgn, Gadd45b, or Sema4C enhanced PCM survival. ∗p < 0.05, ∗∗p < 0.01 versus oligo (n = 4). (B) The transfected PCMs were cultured in basal medium with 0.5 μM doxorubicin for 24 h and subjected to Annexin staining followed by flow cytometry. Silencing circNlgn, Gadd45b, or Sema4C repressed PCM apoptosis. ∗∗p < 0.01 versus oligo (n = 4). (C) PCFs were cultured in basal medium treated with 0.2 μM doxorubicin for 36 h and then transfected with siRNAs. After 48 h, the cells were harvested and subjected to flow cytometry. Silencing circNlgn or RAD50 repressed PCF cycle entry. ∗∗p < 0.01 versus oligo (n = 4). (D) Cell proliferation assays showed that silencing circNlgn or RAD50 repressed PCF proliferation. ∗∗p < 0.01 versus oligo (n = 4). (E) PCR showed that circNlgn, Gadd45b, or Sema4C repressed collagen I (left) and collagen III (right) in the above doxorubicin-treated PCFs. ∗∗p < 0.01 versus oligo (n = 4). We found that transgenic expression of circNlgn mediates doxorubicin-induced cardiofibrosis.…