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Knockdown of RMST Impedes Neuronal Apoptosis and Oxidative Stress in OGD/R-Induced Ischemic Stroke Via Depending on the miR-377/SEMA3A Signal Network

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Abstract

Long non-coding RNAs (lncRNAs) have pivotal roles in regulating ischemic stroke (IS), including lncRNA rhabdomyosarcoma 2-associated transcript (RMST). The purpose of this report is to discover the functional mechanism of RMST. The expression detection of RMST, microRNA-377 (miR-377) and Semaphorin 3A (SEMA3A) was performed by quantitative real-time polymerase chain reaction (qRT-PCR). Oxygen and glucose deprivation/reperfusion (OGD/R) in N2a cells was used to mimic IS environment in vitro. Cell Counting Kit-8 (CCK-8) and flow cytometry were implemented to assess cell viability and apoptosis. Oxidative stress was analyzed via assaying the associated indicators. Dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation (RIP) assays were jointly administrated for binding analysis between targets. SEMA3A protein level was measured using western blot. We found in IS serum samples, RMST was upregulated while miR-377 was downregulated. After the establishment of OGD/R-induced IS model, we found that the decreased RMST abrogated the OGD/R-triggered apoptosis and oxidative stress. Through the target analysis, miR-377 was shown to be sponged by RMST and the effects of RMST knockdown on OGD/R-induced cell injuries were related to miR-377 upregulation. Besides, SEMA3A served as a target gene of miR-377 and the mitigation of miR-377 for ischemic brain damages was achieved by downregulating SEMA3A. What’s more, RMST could regulate SEMA3A by playing the sponge action on miR-377. Collectively, all these findings clarified that RMST repression retarded IS progression in vitro via SEMA3A downregulation by targeting miR-377, which represented a different perspective in the pathological development of IS.

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Data Availability

The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

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Author notes

  1. Lei Zhao and Meng Zhang contribute to this work equally as co-first authors.

Authors and Affiliations

  1. Department of Neurology, Heze Third People’s Hospital, Heze, Shandong, China

    Lei Zhao

  2. Department of Health Management, Zaozhuang Hospital of Traditional Chinese Medicine, Zaozhuang, Shandong, China

    Meng Zhang

  3. Department of General Medicine, Qingdao Central Hospital, Qingdao, Shandong, China

    Fang Yan

  4. Emergency Department, Zaozhuang Hospital of Traditional Chinese Medicine, 2666 Taihang Shan Road, Xuecheng District, Zaozhuang, 277000, Shandong, China

    Yuxia Cong

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  1. Lei Zhao
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  2. Meng Zhang
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Contributions

All authors made substantial contribution to conception and design, acquisition of the data, or analysis and interpretation of the data; take part in drafting the article or revising it critically for important intellectual content; gave final approval of the revision to be published; and agree to be accountable for all aspect of the work.

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Correspondence to Yuxia Cong.

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The authors declare that they have no competing interests.

Ethical Approval

The present study was approved by the ethical review committee of Heze Third People’s Hospital.

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Written informed consent was obtained from all enrolled patients.

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Supplementary Information

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11064_2020_3194_MOESM1_ESM.tif

Supplemental Fig.1. MiR-377 was a potential target of RMST. The luciferase activity was detected by dual-luciferase reporter system after co-transfection of RMST-WT and different miRNAs (miR-881, miR-741, miR-3098, miR-3473g, miR-215, miR-7047, miR-6388, miR-7119, miR-7683, miR-377, miR-6238, miR-205, miR-27a, miR-152, miR-7221) in N2a cells. *P < 0.05. (TIF 472 kb)

11064_2020_3194_MOESM2_ESM.tif

Supplemental Fig.2. SEMA3A was a potential target gene of miR-377. The luciferase activity was detected by dual-luciferase reporter system after co-transfection of miR-377 and each luciferase plasmid (STK35, BEND3, ZFP36I1, RNF38, ANAPC4, KDM6B, YES1, MPRIP, TMX1, WHSC1, SEMA3A, TEAD1, ZFP148, PVR, PCDH10). *P < 0.05. (TIF 444 kb)

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Zhao, L., Zhang, M., Yan, F. et al. Knockdown of RMST Impedes Neuronal Apoptosis and Oxidative Stress in OGD/R-Induced Ischemic Stroke Via Depending on the miR-377/SEMA3A Signal Network. Neurochem Res 46, 584–594 (2021). https://doi.org/10.1007/s11064-020-03194-w

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