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Long non-coding RNA ZFAS1 exerts a protective role to alleviate oxygen and glucose deprivation-mediated injury in ischemic stroke cell model through targeting miR-186-5p/MCL1 axis

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Abstract

In recent years, accumulating articles have revealed that long non-coding RNAs (lncRNAs) play crucial roles in ischemic stroke (IS). A previous study found that lncRNA zinc finger antisense 1 (ZFAS1) was down-regulated in IS patients compared with healthy controls. However, the precise function of ZFAS1 in IS and its associated mechanism remain unclear. Cell viability was assessed by cell counting kit-8 (CCK8) assay. Cell apoptosis was analyzed by flow cytometry. Western blot assay and quantitative real-time polymerase chain reaction (qRT-PCR) were conducted to measure protein and RNA expression. The interaction between microRNA-186-5p (miR-186-5p) and ZFAS1 or MCL1 apoptosis regulator, BCL2 family member (MCL1) was confirmed by dual-luciferase reporter assay, RNA-pull down assay and RNA immunoprecipitation (RIP) assay. IS cell model was established through exposing N2a cells to oxygen and glucose deprivation (OGD). OGD exposure restrained the viability and induced the apoptosis of N2a cells. OGD exposure down-regulated the expression of ZFAS1 and up-regulated the level of miR-186-5p in a time-dependent manner. ZFAS1 overexpression alleviated OGD-mediated injury in IS cell model. MiR-186-5p was identified as a direct target of ZFAS1, and OGD-induced injury in IS cell model was attenuated by the silence of miR-186-5p. MiR-186-5p interacted with the 3′ untranslated region (3′UTR) of MCL1 messenger RNA (mRNA). ZFAS1 positively regulated MCL1 mRNA expression by sequestering miR-186-5p in N2a cells. ZFAS1 overexpression-mediated protective effects in IS cell model were partly overturned by the overexpression of miR-186-5p. MCL1 silencing partly counteracted the protective effects mediated by miR-186-5p silencing in IS cell model. In conclusion, ZFAS1 overexpression exerted a protective role in IS cell model to attenuate OGD-induced injury through targeting miR-186-5p/MCL1 axis. ZFAS1/miR-186-5p/MCL1 signaling might be a novel diagnostic marker and promising treatment target for IS patients.

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Data availability

All data generated or analyzed during this study are included in this article.

Abbreviations

lncRNA:

Long non-coding RNA

miRNA:

MicroRNA

ZFAS1:

Zinc finger antisense 1

IS:

Ischemic stroke

MCL1:

MCL1 apoptosis regulator, BCL2 family member

OGD:

Oxygen and glucose deprivation

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Funding

This work was funded by Yancheng Development Project of Medical Science and Technology in 2020 (YK2020061)

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Authors and Affiliations

  1. Jiangsu Vocational College of Medicine, Jinhua Garden, Chaosheng Road, Tinghu District, Yancheng, 224005, Jiangsu, China

    Bin Shen, Hongwei Wang & Shiyi He

  2. Hubei University of Chinese Medicine, Wuhan, 430065, Hubei, China

    Lan Wang

  3. Wuchang University of Technology, Wuhan, 430223, Hubei, China

    Yuejun Xu

Authors
  1. Bin Shen
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  2. Lan Wang
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  3. Yuejun Xu
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  4. Hongwei Wang
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  5. Shiyi He
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Correspondence to Bin Shen.

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10616_2021_481_MOESM1_ESM.tif

Supplementary file1 ZFAS1 overexpression rescues the expression of cyclin D1 in OGD-induced N2a cells. (A) The protein expression of cyclin D1 was determined in N2a cells in the following four groups by Western blot assay: control, OGD, OGD + vector and OGD + ZFAS1. (B) The level of cyclin D1 was measured in N2a cells in the following five groups by Western blot assay: control, OGD, OGD + vector + miR-NC, OGD + ZFAS1 + miR-NC and OGD + ZFAS1 + miR-186-5p. *P < 0.05, **P < 0.01. (TIFF 452 kb)

10616_2021_481_MOESM2_ESM.tif

Supplementary file2 The silence of MCL1 counteracts miR-186-5p knockdown-induced effects in N2a cells upon OGD stimulation better than the silence of IGF-1. (A and B) The mRNA and protein expression of IGF-1 was determined in N2a cells transfected with anti-NC + si-NC, anti-miR-186-5p + si-NC and anti-miR-186-5p + si-IGF-1. (C-G) N2a cells were divided into six groups: control group, OGD group, OGD + anti-NC + si-NC group, OGD + anti-miR-186-5p + si-NC group, OGD + anti-miR-186-5p + si-MCL1 group and OGD + anti-miR-186-5p + si-IGF-1 group. (C) CCK8 assay was applied to analyze cell viability. (D) Flow cytometry was employed to assess cell apoptosis rate. (E and F) Western blot assay was conducted to determine the expression of Bax, Bcl-2 and cleaved caspase-3 in N2a cells. (G) The level of cyclin D1 was determined in N2a cells by Western blot assay. *P < 0.05, **P < 0.01. (TIFF 1159 kb)

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Shen, B., Wang, L., Xu, Y. et al. Long non-coding RNA ZFAS1 exerts a protective role to alleviate oxygen and glucose deprivation-mediated injury in ischemic stroke cell model through targeting miR-186-5p/MCL1 axis. Cytotechnology 73, 605–617 (2021). https://doi.org/10.1007/s10616-021-00481-4

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