Volume 359, January 2023, 114266Author links open overlay panelAbstractBackground

Mitochondrial autophagy, the elimination of damaged mitochondria through autophagy, contributes to neuron survival in cerebral ischemia. Long non-coding RNAs (lncRNAs)/microRNAs (miRNAs)/mRNAs are important regulatory networks implicated in various biological processes, including cerebral ischemia-reperfusion (I/R) injury. Therefore, this work clarifies a novel RGD1564534-mediated regulatory network on mitochondrial autophagy in cerebral I/R injury.

Methods

Differentially expressed lncRNAs in cerebral I/R injury were predicted by bioinformatics analysis. Expression of RGD1564534 was examined in the established middle cerebral artery occlusion (MCAO) rats and oxygen glucose deprivation/reoxygenation (OGD/R)-exposed neurons. We conducted luciferase activity, RNA pull-down and RIP assays to illustrate the interaction among RGD1564534, miR-101a-3p and Dusp1. Gain- or loss-of-function approaches were used to manipulate RGD1564534 and Dusp1 expression. The mechanism of RGD1564534 in cerebral I/R injury was evaluated both in vivo and in vitro.

Results

RGD1564534 was poorly expressed in the MCAO rats and OGD/R-treated cells, while its high expression attenuated nerve damage, cognitive dysfunction, brain white matter and small vessel damage in MCAO rats. In addition, RGD1564534 promoted mitochondrial autophagy and inhibited NLRP3 inflammasome activity. RGD1564534 competitively bound to miR-101a-3p and attenuated its binding to Dusp1, increasing the expression of Dusp1 in neurons. By this mechanism, RGD1564534 enhanced mitochondrial autophagy, reduced NLRP3 inflammasome activity and suppressed the neuron apoptosis induced by OGD/R.

Conclusion

Altogether, RGD1564534 elevates the expression of Dusp1 by competitively binding to miR-101a-3p, which facilitates mitochondrial autophagy-mediated inactivation of NLRP3 inflammasome and thus retards cerebral I/R injury.

Section snippetsBackground

Stroke has been a great burden in China in recent years (Chao et al., 2021) and its prevalence remains particularly high in rural areas (Wang et al., 2017, (Jia et al., 2010), showing a north-to-south gradient in China (Wu et al., 2019). Cerebral ischemia-reperfusion (I/R) injury is a common feature of ischemic stroke, involving the transient loss and the following rapid return of blood flow to the brain (Bai et al., 2018; Wang et al., 2020). NLRP3 inflammasome is activated in the context of

Ethics statement

The study was approved by the Animal Ethics Committee of Shanghai Pudong Hospital, Fudan University Pudong Medical Center and performed in strict accordance with the Guide for the Care and Use of Laboratory animals published by the US National Institutes of Health.

In silico prediction

Rat cerebral I/R injury-related microarray data GSE106931 demonstrated that Dusp1 can activate mitophagy and GSE25556 were retrieved from the Gene Expression Omnibus (GEO) database. GSE106931 was generated on the GPL1355 [Rat230_2]

RGD1564534 underexpression occurred in MCAO rats and OGD/R cells

In order to explore the molecular mechanism of cerebral I/R, differential analysis was conducted on the cerebral I/R-related microarray data GSE106931. Accordingly, 3675 differentially expressed genes were identified in I/R injury (Fig. 1A, B), of which, only RGD1564534 was down-regulated in MCAO rats (Fig. 1C). This specific downregulation of lncRNA RGD1564534 in MCAO rats has attracted our attention.

As shown in Fig. 1D, E, the neurological deficit score and infarct volume of MCAO rats were

Discussion

The present study suggested that RGD1564534 could attenuate cerebral I/R injury in vivo and neuron apoptosis in vitro. The mechanisms of the protective effect of RGD1564534 might be associated with derepressed miR-101a-3p-mediated Dusp1 inhibition, augmented mitochondrial autophagy and alleviated NLRP3 inflammasome activity and neuron damage.

Accumulating lncRNAs have been documented to be downregulated in cerebral I/R injury in vitro and in vivo, such as SNHG4, SNHG16 and Rian (Pan et al., 2020a

Conclusion

In conclusion, our study revealed that RGD1564534 could weaken the binding of miR-101a-3p to Dusp1 and promoted the mitochondrial autophagy, blocked NLRP3 inflammasome activity and slowed neuron damage, ultimately alleviating cerebral I/R injury (Fig. 7). The RGD1564534/miR-101a-3p/Dusp1 regulatory loop may be a new strategy for developing novel treatment for cerebral I/R injury. However, further in vivo rescue experiments are required to evaluate the effect of lentivirus and/or shRNAs-mediated

Ethic approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Funding

This work was supported by grants from Pudong New Area Clinical Plateau Discipline Project (PWYgy2021-03), Research Grant for Special project for health industry of Pudong Municipal Commission of Health committee of Shanghai (PW2019E-5), Science and Technology Commission of Shanghai Municipality (20Y11909700), Clinical Medicine Innovation project of Fudan Zhangjiang, Fudan Zhangjiang Institute, Shanghai, China (No. KP9202102, FDZJ-202102), the Science and Technology Project of Suzhou City of

Author’ contribution

Weijian Fan and Yuanyuan Qin conceived and designed research. Jinyun Tan and Bo Li performed experiments. Yizhi Liu and Jianjie Rong interpreted results of experiments. Weihao Shi analyzed data and prepared figures. Weijian Fan and Bo Yu drafted paper. Yuanyuan Qin and Weihao Shi edited and revised manuscript. All authors read and approved the final manuscript.

Declaration of Competing Interest

The authors declare that they have no conflicts of interest.

Acknowledgements

Not applicable.

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