Circular RNA PVT1 silencing prevents ischemia-reperfusion injury in rat by targeting microRNA-125b and microRNA-200a

https://doi.org/10.1016/j.yjmcc.2021.05.019Get rights and content

Highlights

  • circRNA PVT1 protects the myocardium from myocardial infarction and H/R injury.

  • circPVT1 silencing promotes cell viability and proliferation while reduces apoptosis.

  • circPVT1 functions as a ceRNA to sponge both miR-125b and miR-200a.

Abstract

Circular RNAs (circRNAs) are essential regulators associated with many cardiac conditions, including myocardial infarction (MI). This study aimed to explore circRNA expression during MI development in an animal model and in hypoxia/reoxygenation (H/R)-treated cardiomyocytes. Microarray and real-time quantitative PCR showed that the circRNA PVT1 (circPVT1) was expressed at high levels in MI tissues and H/R-triggered cardiomyocytes. Loss-of-function assays were utilized for examining the influence of circPVT1 on cardiac function and cardiomyocyte properties. Cardiac function was measured by echocardiography at 7 d after MI. Reduced circPVT1 expression significantly decreased MI-triggered myocardial infarct size by 60% and prevented MI-triggered reductions in fractional shortening (%FS) and ejection fraction (EF%). Results of LDH, CCK-8, EdU staining, colony formation assays, and flow cytometry showed that circPVT1 silencing restored cell viability and proliferation while decreased apoptosis. Mechanistic experiments indicated that microRNAs (miR)-125b and miR-200a associated with circPVT1. We demonstrated that circPVT1 functioned as a competitive endogenous RNA (ceRNA) to sponge both miR-125b and miR-200a. Gain-of-function assays showed that miR-125b and miR-200a upregulation partially eliminated the effects of circPVT1 on cardiomyocyte properties. In addition, we found that the previously reported p53/TRAF6, SIRT7, Keap1/Nrf2, and PDCD4 pathways were regulated by the circPVT1/miR-125b/miR-200a axis. In conclusion, our study suggests that circPVT1 protects the myocardium from MI and H/R injury by preventing miR-125b- and miR-200a-mediated apoptotic signaling.

Introduction

Myocardial infarction (MI) predominantly results from coronary heart disease, particularly coronary thrombosis and is also a main cause of cardiac disease-related deaths [1]. MI has recently gained attention in clinical and research settings. In acute MI, blood supply interruption results in sudden myocardium ischemia, causing irreversible damage to the heart [2]. Hypoxic heart failure can occur, which refers to a situation where the blood delivery rate to tissues and organs fails to meet metabolic requirements [3]. This study detected gene expression changes and observed altered biological processes in hypoxia/reoxygenation (H/R)-triggered myocardial cells.

Accumulating studies have identified circular RNAs (circRNAs) in many diverse cell lines [4]. CircRNAs are a large group of endogenous RNAs which are formed through exon skipping or back-splicing events, being made without a polyadenylated tail or 5′ to 3′ polarity. CircRNAs are evolutionarily conserved and many are expressed only in specific cells or at particular development stages [5,6]. Recently, circRNAs have been reported to have critical roles in various pathological conditions, including MI [7]. The circRNA Cdr1as promotes MI by regulating the expression of the microRNA (miRNA) miR-7a [8]. Zhou et al. reported that the circRNA ACR represses autophagy and MI risk through its action on Pink1-mediated FAM65B phosphorylation [9]. Wang et al. reported that the circular RNA MFACR could modulate mitochondrial fission (MF) and apoptosis through direct reduction of miR-652-3p expression, and this reduction, in turn, suppressed MF and cardiomyocyte death through inhibition of MTP18 translation [10].

Several papers have reported that the PVT1 gene, on chromosome 8q24, plays a critical role in cancer by regulating the stability of proteins encoded by oncogenes [11,12]. The PVT1 gene is frequently increased in cancers, such as NSCLC [13]. Circular RNA (circRNA) PVT1 is a circRNA formed from one PVT1 exon that contains 2 long introns on either side and possesses many Alu repeats, likely promoting the occurrence of circRNA PVT1 (circPVT1) [14]. Memczak et al. [15] first identified circPVT1 as circ6 but subsequent papers named it after its host gene PVT1 [[16], [17], [18]]. circPVT1 has been reported to have an oncogenic role in head and neck squamous cell carcinoma (HNSCC) [16] and gastric cancer [17,19]; however, its molecular functions remain unknown.

CircRNAs may serve as competing endogenous RNAs (ceRNAs) to modulate the expression of genes targeted by specific miRNAs [20]. Several miRNAs have been recognized to protect against MI, such as miR-125b [21,22] and miR-200a [23,24]. This study hypothesized that circPVT1 modulates cardiac function, cell proliferation and cell viability after H/R treatment by sponging both miR-125b and miR-200a. To validate this hypothesis, circPVT1 expression was assessed in cell lines and cardiac tissues. Some experiments further confirm the functional correlation between circPVT1 and MI development.

Section snippets

Animals

Healthy male adult BALB/c mice with body weights ranging from 16 to 20 g were provided by Vitalriver Animal Co., Ltd. (Beijing, China). Mice were kept under standard conditions (humidity, 55 ± 5%; temperature, 23 ± 1 °C) and were provided free access to water and food for one week prior to the experiments. All operations were conducted according to the Guide for Laboratory Animal Care and Use (NIH Publication, 8th Edition, 2011). This study was approved by The First Affiliated Hospital of

CircPVT1 expression was increased in the infarcted tissue of MI mice and H/R-exposed cardiomyocytes

To assess circRNAs during MI development, circRNA expression was screened in the infarcted tissue of Sham and MI mice using microarrays. We found that several circRNAs were expressed at significantly higher levels in the MI tissue compared to those of Sham mice (Fig. 1A). Notably, circPVT1 expression was exceptionally increased; therefore, its expression was next evaluated in murine cardiac tissue at day 3 post MI model establishment. qPCR assays confirmed that circPVT1 expression was

Discussion

This study aimed to probe the underlying mechanism of circRNA and miRNA interactions on cardiomyocyte proliferation and apoptosis during MI. Therefore, it was first necessary to identify which RNAs modulated cell viability and apoptosis. Here, the role of circPVT1 in MI was investigated in vivo and in vitro. We showed that the expression of circPVT1 is elevated in cardiac tissue of MI mice and in H/R-exposed cardiomyocytes. Actually, we have isolated the cardiomyocytes, endothelial cells, and

Conclusions

The ceRNA model, including the circRNA-miRNA-mRNA axis, has been investigated in cardiovascular conditions [33], especially MI [34]. The present experiments confirm a role for the circPVT1/miR-125b/−200a pathway in hypoxia-triggered MI cells. These findings are helpful in identifying new molecular pathways in the progression of MI. Our study has some limitations. For example, functional assays in primary cardiomyocytes and further in vivo experiments should be conducted. The mechanism

Funding

This work was supported by the Guangxi Key Research and Development Plan [grant number GuikeAB18126057].

Declaration of Competing Interest

None.

Acknowledgements

None.

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