Knockdown of circROBO2 attenuates acute myocardial infarction through regulating the miR-1184/TRADD axis

Abstract

Background: Studies have found that circular RNAs (circRNAs) play key roles in cardiovascular diseases. However, the function of circROBO2 in acute myocardial infarction (AMI) is unclear. This study aimed to investigate the pathogenesis of circROBO2 in AMI.

Methods: qRT-PCR and Western blot were used to determine the expression levels of circROBO2, miR-1184, and TRADD in AMI and sham-operated mouse models at mRNA and protein level, respectively. The relationship among miR-1184, circROBO2 and TRADD was evaluated by RNA immunoprecipitation (RIP) analysis and luciferase reporter gene analysis. The roles of circROBO2, miR-1184, and TRADD in myocardial cell apoptosis were evaluated using flow cytometry. Ultrasound echocardiography, serum creatine kinase MB (CK-MB) and lactate dehydrogenase (LDH), myocardial infarction area, and myocardial cell apoptosis were measured to examine the effects of circROBO2 on myocardial injury.

Results: The expression levels of miR-1184 were significantly reduced, and the expression levels of circROBO2 and TRADD were significantly increased in MI group. CircROBO2 acted as a sponge for miR-1184 by upregulating the expression of TRADD. In addition, overexpression of miR-1184 enhanced the protective effect of knockdown of circROBO2 by partially inhibiting the expression of TRADD in vivo and in vitro.

Conclusion: Knockdown of circROBO2 reduced the apoptosis of cardiomyocytes by increasing the expression levels of miR-1184, which in turn decreased the expression levels of TRADD in the myocardium post-MI.

Keywords: Acute myocardial infarction; Apoptosis; TRADD; circROBO2; miR-1184.

Fig. 1

Ischemic injury induced the expression of circROBO2. a qRT-PCR to detect the relative expression levels of circROBO2 or ROBO2 mRNA in myocardium of MI and sham operation groups. b qRT-PCR to detect hypoxic treatment expression levels of circROBO2 or ROBO2 mRNA in primary cardiomyocytes. Experiment was repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001. c HE&RNA ISH of circROBO2 in the infarct region, infarct border and the remote region from infarct, respectively. Scale bar: 30 µm. The green fluorescence signal indicated circROBO2. d HE&IHC of Capaspe3 in the infarct region, infarct border and the remote region from infarct, respectively. Scale bar: 30 µm

Fig. 2

CircROBO2 played a biological role through miR-1184. a Circ Interactome predicted the putative targeting sites for circROBO2 and miR-1184. b Luciferase activity analysis. c The RIP method was used in primary cardiomyocytes. Determine the degree of enrichment of circROBO2 and miR-1184 RNA in the IP complex. Anti-immunoglobulin G (IgG) was used as a control. d Knockdown or overexpression of circROBO2 or miR-1184 in primary cardiomyocytes. e The expression levels of miR-1184 in ischemic myocardium and normal tissues. f The expression levels of miR-1184 in myocardial cells of mice exposed to normoxic or hypoxic conditions. All above the experiments were repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

CircROBO2 up-regulated the expression of TRADD by sponging miR-1184. a Putative binding sites for miR-1184 and TRADD3′-UTR. b Analysis of luciferase activity of PGL-miR-1184 in HEK293 cells by co-transfecting withTRADD-3′-UTR-WT or TRADD-3′-UTR-Mut vector. The experiments were repeated three times. P < 0.05, **P < 0.01, ***P < 0.001. c TRADD mRNA expression levels were detected by co-transfecting miR-1184 mimics or miR-NC, circROBO2 vector or control vector in primary cardiomyocytes. The experiments were repeated three times. P < 0.05, **P < 0.01, ***P < 0.001. d Expression levels of TRADD protein in primary myocardial cells after co-transfecting with miR-1184 mimics, miR-NC + circROBO2 vector or miR-1184 + circROBO2. The experiments were repeated three times. e IHC analysis of caspase3 in myocardium form sham and MI group. f Expression levels of TRADD protein in cardiomyocytes of mice exposed to normoxic or hypoxic conditions (12, 24 and 48 h). There mice were used for this experiment. * vs control group, # vs circROBO2 group. **P < 0.01, ^##P < 0.01

Fig. 4

Biological effects of circROBO2 in hypoxic cardiomyocytes in vitro. a, c Cell viability analysis of mouse cardiomyocytes. Cardiomyocytes were exposed to normoxic or hypoxia combined with transfecting with si-NC, si-circROBO2 + miR-1184 mimics, si-circROBO2 + miR-1184 or si-circROBO2 + si-TRADD. b, d Cell dysfunction of mouse cardiomyocytes exposed to normoxic or hypoxia combined with transfecting with si-NC, si-circROBO2 + miR-1184 mimics, si-circROBO2 + miR-1184 or si-circROBO2 + si-TRADD. All above the experiments were repeated three times. * vs normoxia control group, # vs si-NC group, & vs si-circROBO2 group. ***P < 0.001, ^##P < 0.01 and ^&P < 0.05

Fig. 5

Effects of overexpression of circROBO2 on myocardial function. a Relative expression levels of circROBO2, miR-1184 and TRADD mRNA in mice group of sham, MI, si-circROBO2 transfection, miR-1184 transfection and miR-1184 + circROBO2 transfection. b TRADD protein expression levels. LVEF (c), LVFS (d), Expression of LVESd (e) and LVEDd (f) in Sham, MI, si-circROBO2, miR-1184, and si-circROBO2 + miR-1184 groups. Myocardial cells were from three individual mice. * vs normoxic control group, # vs NC group and vs circROBO2 group. ***P < 0.001, ^##P < 0.01 and ^&P < 0.05

Fig. 6

The effects of knockdown of circROBO2 on myocardial injury. Sham, MI, si-circROBO2, miR-1184 and si-circROBO2 + miR -1184 mice serum CK-MB (a) and LDH (b) and myocardial infarction size (c) were detected and compared. All above the experiments were repeated 3 times. * vs normoxia control group, # vs si-NC group, &vs si-circROBO2 group. ***P < 0.001, ^##P < 0.01 and ^&P < 0.05. d HE staining and TUNEL/nkx2.5 co-staining to detect the apoptosis of mouse cardiomyocytes. The red signaling indicate TUNEL, green signaling indicate nkx2.5, Scale bar: 100 μm