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. 2021 Apr 30;30(2):170-182.
doi: 10.5607/en20046.

Upregulation of miR-20b Protects Against Cerebral Ischemic Stroke by Targeting Thioredoxin Interacting Protein (TXNIP)

Dejiang Yang  1 , Yu Tan  1 , Huanhuan Li  1 , Xiaowei Zhang  1 , Xinming Li  1 , Feng Zhou  2
Affiliations

Upregulation of miR-20b Protects Against Cerebral Ischemic Stroke by Targeting Thioredoxin Interacting Protein (TXNIP)

Dejiang Yang et al. Exp Neurobiol. .

Abstract

Dysregulation of microRNAs (miRNAs) is involved in abnormal development and pathophysiology in the brain. Although miR-20b plays essential roles in various human diseases, its function in cerebral ischemic stroke remains unclear. A cell model of oxygen glucose deprivation/reoxygenation (OGD/R) and A rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) were constructed. qRT-PCR and western blot were used to evaluate the expression of miR-20b and TXNIP. Cell viability was detected by MTT assay, and cell apoptosis was evaluated by flow cytometry. Targetscan and Starbase were used to predict the potential targets of miR-20b. Luciferase reporter assay was applied to determine the interaction between miR-20b and TXNIP. Rescue experiments were conducted to confirm the functions of miR-20b/TXNIP axis in cerebral ischemic stroke. MiR-20b was significantly downregulated after I/R both in vitro and in vivo. Upregulation of miR-20b inhibited OGD/R-induced neurons apoptosis and attenuated ischemic brain injury in rat model. Bioinformatic prediction suggested that TXNIP might be a target of miR-20b, and luciferase reporter assay revealed that miR-20b negatively regulated TXNIP expression by directly binding to the 3'-UTR of TXNIP. Downregulation of TXNIP inhibited OGD/R-induced neurons apoptosis in vitro and ischemic brain injury in vivo. Rescue experiments indicated that downregulation of TXNIP effectively reversed the effect of miR-20b inhibitor in neurons apoptosis after OGD/R-treatment and ischemic brain injury in a mouse model after MCAO/R-treatment. Our study demonstrated that upregulation of miR-20b protected the brain from ischemic brain injury by targeting TXNIP, extending our understanding of miRNAs in cerebral ischemic stroke.

Keywords: Apoptosis; Cerebral ischemic stroke; Ischemic brain injury; TXNIP; miR-20b.

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Figures

Fig. 1
Fig. 1
miR-20b was significantly downregulated after I/R both in vitro and in vivo. (A-C) Neurons were treated with OGD/R (2 h/24 h). (A) Cell viability was evaluated by MTT assay. (C) Apoptotic rate was measured by flow cytometry. (C) The mRNA level of miR-20b was detected by qRT-PCR. (D-F) Rats were treated with MCAO/R. (D) Neurobehavioral scores. (E) The representative images of brains tissues and infarct volume. (F) The mRNA level of miR-20b in brain tissues was evaluated by qRT-PCR. **p<0.01.
Fig. 2
Fig. 2
Upregulation of miR-20b protected neurons against OGD/R-induced injury in vitro. Neurons were transfected with miR-20b mimics, miR-NC, miR-20b inhibitor, or inhibitor NC, and then then treated with OGD/R. (A) The mRNA level of miR-20b was detected by qRT-PCR. (B) Cell viability was evaluated by MTT assay. (C) Apoptotic rate was detected by flow cytometry. *p<0.05, **p<0.01.
Fig. 3
Fig. 3
Upregulation of miR-20b attenuated ischemic brain injury in vivo. MiR-20b mimic, miR-20b inhibitor and negative controls were injected into the cerebral cortex of rats, and rats were then treated with MCAO/R. (A) The mRNA level of miR-20b in brain tissues of rats was evaluated by qRT-PCR. (B) Neurobehavioral scores. (C) The representative images of brains tissues and infarct volume. (D) The effect of miR-20b on brain edema after cerebral ischemia. (E) Immunohistochemical staining of cleaved caspase-3 positive cells in cortical neuron (×200 magnification, scale bar=100μm). (F) Quantitative analysis of immunohistochemical staining. *p<0.05, **p<0.01.
Fig. 4
Fig. 4
TXNIP was a target of miR-20b. (A) The putative binding sites between miR-20b and TXNIP were predicted by Targetscan and Starbase databases. (B) The WT or MUT 3’-UTR of TXNIP both in site 1 and site 2 was cloned into pmirGLO reporter vector, and co-transfected with miR-20b mimics or miR-NC into neurons. The relative luciferase activity was detected by dual luciferase reporter system. (C, D) Neurons were transfected with miR-20b, miR-NC, miR-20b inhibitor, or inhibitor NC, and treated with OGD/R. The expression of TXNIP was evaluated by qRT-PCR (C) and western blot (D). **p<0.01.
Fig. 5
Fig. 5
Downregulation of TXNIP inhibited OGD/R-induced neurons apoptosis in vitro and ischemic brain injury in vivo. (A and B) Neurons were transfected with sh-TXNIP or sh-NC. The expression of TXNIP was detected by qRT-PCR (A) and western blot (B). (C, D) Neurons were transfected with sh-TXNIP or sh-NC, and then treated with OGD/R. (C) Cell viability was evaluated by MTT assay. (D) Apoptotic rate was detected by flow cytometry. *p<0.05, **p<0.01. (E-H) sh-TXNIP or sh-NC was injected into the cerebral cortex of rats, and rats were then treated with MCAO/R. The expression of TXNIP in ischemic brain was evaluated by qRT-PCR (E) and western blot (F). (G) Neurobehavioral scores. (H) The representative images of brains tissues and infarct volume. *p<0.05, **p<0.01.
Fig. 6
Fig. 6
Upregulation of miR-20b attenuated OGD/R-induced neurons apoptosis in vitro and ischemic brain injury through targeting TXNIP. (A-C) Neurons were transfected with miR-20b mimics, miR-20b inhibitor, or co-transfected with miR-20b mimics and sh-TXNIP, or miR-20b inhibitor and sh-TXNIP. Then cells were treated with OGD/R. (A) The expression of TXNIP was evaluated by western blot. (B) Cell viability was detected by MTT assay. (C) Apoptotic rate was measured by flow cytometry. *p<0.05, **p<0.01. (D) Rats were cortical injected with miR-20b mimics, miR-20b inhibitor, or co-injection with miR-20b mimics and sh-TXNIP, or miR-20b inhibitor and sh-TXNIP. Then rats were treated with MCAO/R. The representative images of brains tissues and infarct volume. *p<0.05, **p<0.01.
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