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.

中文翻译：

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环状 RNA TLK1 通过 TNF 信号通路靶向 miR-214/RIPK1 加剧心肌缺血/再灌注损伤。

目的

心肌缺血/再灌注损伤 (IRI) 诱导心肌细胞死亡并导致心脏功能丧失。环状 RNA (circRNA) 在调节心肌 IRI 方面越来越受到关注。本研究旨在探讨circTLK1在心肌IRI发病机制中的作用及其确切机制。

方法

心肌 IRI 是在小鼠中开发的，通过测量血流动力学参数和血清心肌酶的活性来评估心脏功能。进行 HE 和 TTC 染色以评估梗死面积。使用 qRT-PCR 分析研究了 circTLK1 和 miR-214 的表达模式。circTLK1、miR-214 或 RIPK 的基因表达通过转染它们的过表达或敲低载体而改变。通过TUNEL染色和Caspase-3活性分析评估心肌细胞的凋亡。通过蛋白质印迹测定凋亡相关标志物 Bcl-2、Bax 和 caspase3，以及 TNF-α 信号。circTLK1/miR-214 和 miR-214/RIPK1 的相互作用通过荧光素酶报告基因分析得到验证。RNA免疫沉淀（RIP）进一步确定circTLK1/miR-214的直接结合。在体内。

结果

circTLK1 是在心肌 IRI 小鼠模型中发现的上调 circRNA。沉默circTLK1的小鼠显着减轻了受损的心脏功能指标并减少了梗死面积，从而减轻了心肌IRI的发病机制。circTLK1 的敲低显着降低了心肌细胞的凋亡，这是由凋亡相关蛋白决定的。miR-214 被确定为一种下游效应器，可逆转 circTLK1 介导的心肌 IRI 损伤效应。miR-214 可以通过与其'3'-UTR 结合直接靶向 RIPK1。RIPK1的过表达导致心脏功能指标受损、梗死面积增加和细胞凋亡，从而消除了miR-214的保护作用。TNF信号通路被证明参与心肌IRI中的circTLK1/miR-214/RIPK1调控网络。

结论

总之，我们的研究揭示了一种上调的 circRNA，circTLK1，可以通过靶向 miR-214/RIPK1 介导的 TNF 信号通路加剧心肌 IRI，这可能为治疗提供治疗靶点。