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CircRNA circ-NNT mediates myocardial ischemia/reperfusion injury through activating pyroptosis by sponging miR-33a-5p and regulating USP46 expression.

ABSTRACT: Pyroptosis has been implicated in the pathophysiology of myocardial infarction (MI) in rodents, but its contribution to reperfusion injury in MI patients is unclear. Here, we evaluated pyroptosis in MI patients in vitro and in vivo models of myocardial ischemia/reperfusion (I/R) injury. We also investigated the molecular mechanisms that regulate pyroptosis and myocardial I/R injury in these in vitro and in vivo models. The study showed that MI patients exhibited elevated serum concentrations of the pyroptosis-related pro-inflammatory cytokines IL-1β and IL-18. Increased levels of IL-1β and IL-18 as well as the pyroptosis-related inflammatory caspases (caspase-1 and 11) were detected in cultured cardiomyocytes after anoxia/reoxygenation (A/R) and in cardiac tissues after I/R. Circ-NNT and USP46 were upregulated while miR-33a-5p was downregulated in MI patients, as well as in cultured cardiomyocytes after A/R and cardiac tissues after I/R. Circ-NNT or USP46 knockdown or miR-33a-5p overexpression inhibited the expression of pro-caspase-1, cleaved caspase-1, pro-caspase-11, cleaved caspase-11, IL-1β, and IL-18 in A/R cardiomyocytes and attenuated myocardial infarction in I/R mice. The results from luciferase reporter assays and gene overexpression/knockdown studies indicated that miR-33a-5p directly targets USP46, and circ-NNT regulates USP46 by acting as a miR-33a-5p sponge. Direct association between circ-NNT and miR-33a-5p in cardiomyocytes was confirmed by pull-down assays. In summary, pyroptosis is activated during myocardial I/R and contributes to reperfusion injury. Circ-NNT promotes pyroptosis and myocardial I/R injury by acting as a miR-33a-5p sponge to regulate USP46. This circ-NNT→miR-33a-5p→USP46 signaling axis may serve as a potential target for the development of cardio-protective agents to improve the clinical outcome of reperfusion therapy.

SUBMITTER: Ye X

PROVIDER: S-EPMC8630116 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Stroke and subsequent cerebral ischemia/reperfusion (I/R) injury is a frequently occurring disease that can have serious consequences in the absence of timely intervention. Circular RNAs (circRNAs) in association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression. However, whether circRNAs have a role in cerebral I/R injury pathogenesis, especially soon after onset, is unclear. In this study, we used the SD rat middle cerebral artery occlusion (MCAO) model of stroke to examine the role of circRNAs in cerebral I/R injury. We used high-throughput sequencing (HTS) to compare the expression levels of circRNAs in cerebral cortex tissue from MCAO rats during the occlusion-reperfusion latency period 3 hours after I/R injury with those in control cerebral cortices. Our sequencing results revealed that expression levels of 44 circRNAs were significantly altered after I/R, with 16 and 28 circRNAs showing significant up- and down-regulation, respectively, relative to levels in control cortex. We extended these results in vitro in primary cultured neuron cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R) using qRT-PCR to show that levels of circ-camk4 were increased in OGD/R neurons relative to control neurons. Bioinformatics analyses predicted that several miRNAs could be associated with circ-camk4 and this prediction was confirmed in a RNA pull-down assay. KEGG analysis to predict pathways that involve circ-camk4 included the glutamatergic synapse pathway, MAPK signaling pathway, and apoptosis signaling pathways, all of which are known to be involved in brain injury after I/R. Our results also demonstrate that levels of the human homolog to circ-camk4 (hsa-circ-camk4) are elevated in SH-SY5Y cells exposed to OGD/R treatment. Overexpression of hsa-circ-camk4 in SH-SY5Y cells significantly increased the rate of cell death after OGD/R, suggesting that circ-camk4 may play a key role in progression of cerebral I/R injury.

Project description:Long non‑coding RNAs (lncRNAs) have been revealed to have a marked effect in cardiovascular diseases, including during cardiac development, cardiac hypertrophy, myocardial fibrosis and myocardial ischemic injury. The mechanism of myocardial ischemia‑reperfusion injury (MIRI) is very complicated. Although studies have confirmed that lncRNAs are involved, the specific mechanism remains largely unknown. The lncRNA growth arrest specific 5 (GAS5) is known as a regulator of a number of diseases, including certain cancer types. The present study aimed to investigate the function of lncRNA GAS5 in MIRI. The present study reported that the expression of lncRNA GAS5 in H9c2 cells treated with anoxia and reoxygenation was significantly upregulated compared with the control group (P<0.05). Similarly, the expression of lncRNA GAS5 in myocardial tissue obtained from rats treated with MIRI was significantly upregulated compared with the untreated controls (P<0.05). Silencing of lncRNA GAS5 was able to attenuate myocardial damage, as cell viability increased and the apoptosis rate decreased. Classical apoptotic proteins involved in MIRI, including B‑cell lymphoma 2, Bcl‑2‑associated X protein and cleaved caspase‑3, also exhibited the same trend. At the same time, when lncRNA GAS5 was silenced, microRNA (miR)‑532‑5p, which was originally expressed at the stage of injury, was upregulated. The luciferase reporter assay results indicated that the lncRNA GAS5 functioned as a molecular sponge of miR‑532‑5p. The gain‑ and loss‑of‑function analysis of miR‑532‑5p indicated that it was involved in the regulation of MIRI; the trend of results following its overexpression was also consistent with the trend observed following the silencing of lncRNA GAS5. Notably, the protective effect of lncRNA GAS5 silencing on cells was attenuated by miR‑532‑5p inhibition. Phosphatase and tensin homolog was revealed to be a key target gene for the function of lncRNA GAS5, and its regulation was achieved via binding to miR‑532‑5p. In other words, silencing lncRNA GAS5 ultimately promoted the activation of the phosphoinositide‑3‑kinase (PI3K)/protein kinase B pathway (AKT) to reduce myocardial damage. Therefore, lncRNA GAS5 was able to regulate MIRI through the PI3K/AKT apoptosis pathway by sponging miR‑532‑5p.

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CircRNA circ-NNT mediates myocardial ischemia/reperfusion injury through activating pyroptosis by sponging miR-33a-5p and regulating USP46 expression.

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