Abstract

Background

Cardiomyocytes apoptosis is the basic pathological process of myocardial ischaemia/reperfusion (MI/R) injury, so inhibiting apoptosis of cardiomyocytes can effectively improve MI/R injury. Long non-coding RNA colorectal neoplasia differentially expressed (lncRNA CRNDE) can inhibit cell apoptosis, but its specific role in MI/R injury has not been studied. The aim of this study is to explore the specific effect of lncRNA CRNDE on cardiomyocytes apoptosis.

Methods

MI/R model in vivo and hypoxia/re-oxygenation (H/R) model in vitro were constructed. Apoptotic levels were assessed by TUNEL staining assay. QRT-PCR was used to validate lncRNA CRNDE level in myocardial tissues and HL-1 cells. The protein expressions of YAP1, Bcl-2 and cleaved caspase-3 were detected by western blot analysis. Flow cytometry was used to determine the apoptosis rate of cardiomyocytes. RIP assay was used to detect the interaction between lncRNA CRNDE and YAP1.

Results

The extent of cardiomyocytes apoptosis was significantly increased, and the levels of lncRNA CRNDE, YAP1 and Bcl-2 were down-regulated, while cleaved caspase-3 expression was up-regulated in MI/R mice and H/R-treated HL-1 cells. The expressions of YAP1 and Bcl-2 were decreased, while the expression of cleaved caspase-3 was increased after the knockdown of lncRNA CRNDE. Furthermore, lncRNA CRNDE could bind to YAP1 and regulated the protein level of YAP1 by ubiquitination and proteasomal degradation pathway. After transfection of Si-YAP1 in the H/R-treated HL-1 cells transfected with pc-DNA CRNDE, the protein level of Bcl-2 was decreased, while cleaved caspase-3 expression and the apoptosis rate were increased.

Conclusion

Our study suggested that lncRNA CRNDE could regulate YAP1 level by ubiquitination and proteasomal degradation pathway, thus inhibiting cardiomyocytes apoptosis in MI/R injury.

中文翻译：

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LncRNA CRNDE通过YAP1抑制心肌缺血/再灌注损伤中的心肌细胞凋亡

摘要

背景

心肌细胞凋亡是心肌缺血/再灌注（MI/R）损伤的基本病理过程，因此抑制心肌细胞凋亡可有效改善MI/R损伤。长链非编码RNA大肠肿瘤差异表达（lncRNA CRNDE）可抑制细胞凋亡，但其在MI/R损伤中的具体作用尚未研究。本研究旨在探讨lncRNA CRNDE对心肌细胞凋亡的特异性作用。

方法

构建了体内MI/R模型和体外缺氧/复氧(H/R)模型。通过 TUNEL 染色测定评估细胞凋亡水平。QRT-PCR 用于验证心肌组织和 HL-1 细胞中的 lncRNA CRNDE 水平。通过蛋白质印迹分析检测YAP1、Bcl-2和cleaved caspase-3的蛋白表达。流式细胞仪用于测定心肌细胞的凋亡率。RIP 检测用于检测 lncRNA CRNDE 和 YAP1 之间的相互作用。

结果

MI/R 小鼠和 H/R 处理的 HL-1 心肌细胞凋亡程度显着增加，lncRNA CRNDE、YAP1 和 Bcl-2 水平下调，而 cleaved caspase-3 表达上调细胞。敲低lncRNA CRNDE后，YAP1和Bcl-2的表达降低，而cleaved caspase-3的表达增加。此外，lncRNA CRNDE 可以与 YAP1 结合并通过泛素化和蛋白酶体降解途径调节 YAP1 的蛋白水平。在转染pc-DNA CRNDE的H/R处理的HL-1细胞中转染Si-YAP1后，Bcl-2蛋白水平降低，而cleaved caspase-3表达和细胞凋亡率增加。

结论

我们的研究表明lncRNA CRNDE可以通过泛素化和蛋白酶体降解途径调节YAP1水平，从而抑制MI/R损伤中的心肌细胞凋亡。