The circular RNA TLK1 exacerbates myocardial ischemia/reperfusion injury via targeting miR-214/RIPK1 through TNF signaling pathway.

Purpose: Myocardial ischemia/reperfusion injury (IRI) induces cardiomyocytes death and leads to loss of cardiac function. Circular RNAs (circRNA) have gain increasing interests in modulating myocardial IRI. In this study, we aim to investigate the role and exact mechanism of circTLK1 in the pathogenesis of myocardial IRI.

Methods: Myocardial IRI was developed in mice with measuring hemodynamic parameters and the activity of serum myocardial enzymes to evaluate cardiac function. HE and TTC staining were performed to assess infarct area. Expression patterns of circTLK1 and miR-214 were investigated using qRT-PCR assay. Gene expression of circTLK1, miR-214 or RIPK was altered by transfecting with their overexpression or knockdown vectors. The apoptosis of cardimyocytes was assessed by TUNEL staining and Caspase-3 activity analysis. Apoptosis-related markers Bcl-2, Bax, and caspase3, as well as TNF-α signals were determined by western blotting. The interactions of circTLK1/miR-214 and miR-214/RIPK1 were verified using luciferase reporter assay. RNA immunoprecipitation (RIP) was subjected to further definite the direct binding of circTLK1/miR-214. The regulatory network of circTLK1/miR-214/RIPK1 was further validated in vivo.

Results: circTLK1 was an up-regulated circRNA found in a myocardial IRI mouse model. Mice with silencing circTLK1 significantly alleviated the impaired cardiac function indexes and decreased infarct area, thus attenuating the pathogenesis of myocardial IRI. Knockdown of circTLK1 dramatically decreased cardiomyocytes apoptosis, which was determined by apoptosis-related proteins. miR-214 was identified as a downstream effector to reverse circTLK1-mediated damage effects in myocardial IRI. miR-214 could directly target RIPK1 via binding to its' 3'-UTR. Overexpression of RIPK1 led to impaired cardiac function indexes, increased infarct area, and cell apoptosis, which abolished the protective effects of miR-214. The TNF signaling pathway was demonstrated to be involved in the circTLK1/miR-214/RIPK1 regulatory network in myocardial IRI.

Conclusion: Taken together, our study revealed an up-regulated circRNA, circTLK1, could exacerbate myocardial IRI via targeting miR-214/RIPK1-mediated TNF signaling pathway, which may provide therapeutic targets for treatment.