A circular transcript of ncx1 gene mediates ischemic myocardial injury by targeting miR-133a-3p

Abstract

Non-coding RNAs (ncRNAs) are considered major players in physiological and pathological processes based on their versatile regulatory roles in different diseases including cardiovascular disease. Circular RNAs (circRNAs), a newly discovered class of RNAs, constitute a substantial fraction of the mammalian transcriptome and are abundantly expressed in the cardiovascular system. However, the regulatory functions of these circRNAs in ischemic cardiac disease remain largely unknown. Here, we investigated the role of a circRNA transcribed from the sodium/calcium exchanger 1 (ncx1) gene, named circNCX1, in oxidative stress-induced cardiomyocyte apoptosis during ischemic myocardial injury. Methods: Divergent polymerase chain reaction (PCR) was conducted to amplify the circRNA. The circular structure of circNCX1 was verified by Sanger sequencing and RNase R digestion. The subcellular localization of circNCX1 was detected by fluorescence in situ hybridization (FISH). To test the expression pattern and function of circNCX1 during oxidative stress, H9c2 cells and neonatal rat cardiomyocytes were treated with H₂O₂ or hypoxia-reoxygenation (H/R). Mechanistically, the interaction of circNCX1 with miRNA was examined by AGO2-IP and RNA pull-down assays. The regulatory role of circNCX1 in target gene expression was tested by western blot and luciferase reporter assays. At the animal level, we constructed a myocardial ischemia-reperfusion (I/R) mouse model to analyze the effect of circNCX1 on heart function, cardiomyocyte apoptosis and cardiac remodeling. Results: circNCX1 was increased in response to reactive oxygen species (ROS) and promotes cardiomyocyte apoptosis by acting as an endogenous miR-133a-3p sponge. Due to competitive binding of circNCX1 to miR-133a-3p, the suppressive activity of pro-apoptotic gene cell death-inducing protein (CDIP1) by miR-133a-3p was reduced. Knockdown of circNCX1 in murine cardiomyocytes and heart tissues reduced the levels of CDIP1 and attenuated the apoptosis and I/R injury. Conclusions: Our findings reveal a novel regulatory pathway that comprises circNCX1, miR-133a-3p and CDIP1, that is involved in cardiomyocyte apoptosis. This pathway may serve as a potential therapeutic avenue for ischemic heart diseases.

Keywords: CDIP1; cardiomyocyte apoptosis; circular RNA; ischemic cardiomyopathy; miR-133a.

Figure 1

Verification of circNCX1. (A) circNCX1 was generated from the 2^nd exon of the ncx1 gene. Sequence analysis of PhyloP showed that circNCX1 is conserved. circNCX1 exists in mouse (B) and rat (C) myocardial tissue. Clear single bands were amplified from the cDNA of mouse and rat myocardial tissue by divergent primers, while they could not be amplified from gDNA. Sanger-Seq validated the head-to-tail junction of mouse (D) and rat (E) circNCX1. (F) RNAs from mouse cardiac tissue were incubated with RNase R or buffer only (Mock). After digestion, the RNAs were purified. The levels of circNCX1, NCX1 mRNA and GAPDH mRNA were analyzed by qRT-PCR. *P < 0.05 versus Mock. n=3. (G) RNAs were obtained from neonatal rat cardiomyocytes and cardiac fibroblasts. The levels of circNCX1 in cardiomyocytes and fibroblasts were analyzed by qRT-PCR. *P < 0.05 versus cardiomyocytes. n=3. (H) circNCX1 identified by specific probes are shown in green. DAPI-stained nuclei are shown in blue. Scale bars: upper, 30 μm; lower, 10 μm.

Figure 2

circNCX1 promotes cardiomyocyte apoptosis. (A) H9c2 cells were treated with 100 μΜ H₂O₂. Total RNA was isolated and reverse-transcribed. The circNCX1 level was analyzed by qRT-PCR. *P < 0.05 versus 0 h. n=3. (B) H9c2 cells were treated with H/R. The circNCX1 level was analyzed by qRT-PCR. *P < 0.05 versus control. n=3. (C) Total RNA of mouse ischemic cardiac tissue was isolated and reverse-transcribed. The circNCX1 level was analyzed by qRT-PCR. *P < 0.05 versus 0 min. n=6. (D) H9c2 cells were transfected with the shRNA vector. The expression level of circNCX1 was analyzed by qRT-PCR. SC: scramble control; ShRNA: shRNA targeting the junction parts of circNCX1. *P < 0.05 versus SC. n=3. (E) H9c2 cells were transfected with the shRNA vector and were treated with 100 μΜ H₂O₂ for 12 h. Cell apoptosis was analyzed by the TUNEL assay. Representative images are shown on the left. The apoptosis rate calculated from three independent experiments is shown on the right. Green, TUNEL-positive nuclei; blue, DAPI-stained nuclei. Scale bars, 50 μm. *P < 0.05 versus SC. n=3. (F) H9c2 cells were transfected with the shRNA vector and treated with H/R. Cell apoptosis was analyzed by the TUNEL assay. *P < 0.05 versus SC. n=3. (G) H9c2 cells were transfected with the circNCX1 expression vector. The expression level of circNCX1 was analyzed by qRT-PCR. EV: empty vector. *P < 0.05 versus EV. n=3. (H) H9c2 cells were transfected with the circNCX1 overexpression vector and were treated with 50 μΜ H₂O₂ for 12 h. Cell apoptosis was detected by the TUNEL assay. Scale bars, 50 μm. *P < 0.05 versus EV. n=3.

Figure 3

circNCX1 interacts with miR-133a-3p. (A) Immunoprecipitation of AGO2 (control, mouse IgG) in H9c2 cells was performed. The associated RNAs were purified. The levels of circNCX1 and GAPDH mRNA were analyzed by qRT-PCR. The relative pellet/ input ratios were calculated. *P < 0.05 versus GAPDH. n=3. (B) circNCX1 contains 8 potential binding sites of miR-133a-3p. (C) Biotin-labeled miR-133a-3p mimics or control mimics were transfected into H9c2 cells. The level of total and streptavidin-captured GAPDH mRNA and circNCX1 were respectively analyzed by qRT-PCR. The relative pellet/input ratios were calculated. Bio-NC: biotin-labeled control mimics. *P < 0.05 versus bio-NC. n=3. (D) The levels of total and pulled-down miR-133a-3p and U6 by circNCX1 probe or control probe were analyzed by qRT-PCR. The relative pellet/input ratios were calculated. Random: scrambled control probes; Circ: circNCX1 probes. *P < 0.05 versus Random. n=3. (E) Immunoprecipitation of AGO2 (control, mouse IgG) in miR-133a-3p or control mimics (NC)-transfected H9c2 cells was performed. The associated RNAs were purified. The levels of circNCX1 and GAPDH mRNA were analyzed by qRT-PCR. The relative pellet/input ratios were calculated. *P < 0.05 versus NC. n=3. (F) circNCX1 identified by specific probes is shown in green. miR-133a-3p is shown in red. DAPI-stained nuclei are shown in blue. Scale bar, 10 μm. (G) H9c2 cells were transfected with the circNCX1 expression vector. The expression level of miR-133a-3p was analyzed by qRT-PCR. n=3. (H) H9c2 cells were transfected with the shRNA vector. The expression level of miR-133a-3p was analyzed by qRT-PCR. n=3. (I) H9c2 cells were treated with 100 μΜ H₂O₂. The expression level of miR-133a-3p was analyzed by qRT-PCR. n=3. (J) The expression level of miR-133a-3p in mouse ischemic cardiac tissue was analyzed by qRT-PCR. n=6.

Figure 4

circNCX1 increases cardiomyocyte apoptosis by inhibiting miR-133a-3p. (A) H9c2 cells were transfected with the miR-133a-3p inhibitor (Inhibitor-133a) or control inhibitor (Inhibitor-nc) and were treated with 50 μM H₂O₂ for 12h. The levels of cleaved (Cl) and full-length (Fl) caspase-3 were analyzed by western blotting. β-Actin was selected as a reference. A representative image is shown at the bottom. The relative protein level calculated by ImageJ from three independent experiments is shown at the top. *P < 0.05 versus inhibitor-nc. (B) H9c2 cells were transfected with miR-133-3p inhibitors and were treated with 50 μM H₂O₂ for 12 h. Cell apoptosis was detected by the TUNEL assay. *P < 0.05 versus Inhibitor-nc. n=3. (C) circNCX1 expression vector and miR-133a-3p mimics (or control mimics) were co-transfected into H9c2 cells. Cells were treated with 50 μM H₂O₂ for 12 h. Cell apoptosis was detected by TUNEL assay. *P < 0.05 versus Mimic-nc. n=3. (D) circNCX1 shRNA vector and miR-133a-3p inhibitors (or control inhibitors) were co-transfected into H9c2 cells. Cells were treated with 100 μM H₂O₂ for 12 h. Cell apoptosis was detected by the TUNEL assay. *P < 0.05 versus inhibitor-nc. n=3.

Figure 5

miR-133a-3p suppresses apoptosis by targeting CDIP1. (A) A conserved binding site of miR-133a-3p exists in the 3' UTR of cdip1. The expression of CDIP1 in 100 μM H₂O₂-treated H9c2 cells (n=3, *P < 0.05 versus 0 h) (B) and ischemic cardiac tissue (n=6, *P < 0.05 versus 0 min) (C) was analyzed by western blotting. (D) H9c2 cells were transfected with miR-133a-3p mimics and were treated with 100 μM H₂O₂ for 12 h. The expression of CDIP1 was analyzed by western blotting. *P < 0.05 versus Mimic-nc. n=3. (E) HEK293 cells were transfected with the pGL3 vector containing the wild type CDIP1 3' UTR or mutant 3' UTR as well as miR-133a-3p mimics or control mimics. The cells were harvested, and luciferase activity was measured. *P < 0.05 versus Mimic-nc. n=3. (F) CDIP1 siRNA or control oligo were transfected into H9c2 cells. Cells were treated with 100 μM H₂O₂ for 12 h. Cell apoptosis was detected by the TUNEL assay. CDIP1-sc: control oligo. *P < 0.05 versus CDIP1-sc. (G) miR-133a-3p inhibitor as well as CDIP1 siRNA or control oligo were co-transfected into H9c2 cells. Cells were treated with 50 μM H₂O₂ for 12 h. Cell apoptosis was detected by the TUNEL assay. *P < 0.05 versus CDIP1-sc.

Figure 6

circNCX1 promotes cardiomyocyte apoptosis by increasing the CDIP1 level. (A) HEK293 cells were co-transfected with the pGL3 vector containing the 3' UTR of CDIP1, miR-133a-3p mimics and circNCX1 expression vector (or empty vector). The cells were harvested and luciferase activity was measured. *P < 0.05 versus EV. n=3. (B) H9c2 cells were transfected with the circNCX1 expression vector and were treated with 50 μM H₂O₂ for 12 h. The expression of CDIP1 was analyzed by western blotting. *P < 0.05 versus EV. n=3. (C) H9c2 cells were transfected with the shRNA expression vector and treated with 100 μM H₂O₂ for 12 h. The expression of CDIP1 was analyzed by western blotting. *P < 0.05 versus circNCX1-sc. n=3. (D) H9c2 cells were co-transfected with the circNCX1 over-expression vector and miR-133a-3p mimics. Cells were treated with 50 μM H₂O₂ for 12 h. The expression of CDIP1 was analyzed by western blotting. *P < 0.05 versus Mimic-nc. n=3. (E) H9c2 cells were co-transfected with the shRNA vector and miR-133a-3p inhibitors. Cells were treated with 100 μM H₂O₂ for 12 h. The expression of CDIP1 was analyzed by western blotting. *P < 0.05 versus Inhibitor-nc. n=3. (F) H9c2 cells were co-transfected with the circNCX1 over-expression vector and CDIP1 siRNAs. Cells were treated with 50 μM H₂O₂ for 12 h. Cell apoptosis was detected by the TUNEL assay. *P < 0.05 versus CDIP1-sc. n=3.

Figure 7

Silencing of circNCX1 in the heart attenuates cardiac ischemia-reperfusion injury. (A) Mouse hearts were infected by shRNA adenovirus. The infected mice were subjected to sham operation or I/R. The expression level of circNCX1 in mouse hearts was analyzed by qRT-PCR. *P < 0.05 versus SC. n=6. (B) The expression level of miR-133a-3p in mouse hearts was analyzed by qRT-PCR. n=6. (C) The expression level of CDIP1 in mouse hearts was analyzed by western blotting. *P < 0.05 versus SC. n=6. (D) Representative images of ventricular myocardium sections from mice exposed to sham operation or I/R. Green, TUNEL-positive nuclei; blue, DAPI-stained nuclei; red, cardiomyocytes labeled with antibody to α-actinin; scale bar, 50 μm. Quantitative analysis of apoptosis is shown at the bottom. *P < 0.05 versus SC. n=6. (E) Infarct sizes (right) and representative images of midventricular myocardial slices (left) from mice exposed to I/R or sham operation. AAR: area at risk; LV: left ventricular area; INF: infarct area. Scale bars, 2 mm. *P < 0.05 versus SC. n = 6. (F) Collagen areas of mice subjected to I/R or sham operation. Scale bars, 20 μm. *P < 0.05 versus SC. n = 6. (G) Echocardiographic analysis of left ventricular dimensions and cardiac function in mice exposed to I/R. LVIDs: systolic left ventricular internal diameters; FS: fractional shortening of the left ventricular diameter; LVEF: left ventricular ejection fraction. *P < 0.05 versus SC. n = 6.