lncRNA WT1-AS attenuates hypoxia/ischemia-induced neuronal injury during cerebral ischemic stroke via miR-186-5p/XIAP axis

Abstract

This study aimed to investigate the role and mechanism of long non-coding RNA (lncRNA) WT1 antisense RNA (WT1-AS) in cerebral ischemic stroke. The Starbase database and dual-luciferase reporter gene assay were used to analyze the interaction between lncRNA WT1 antisense RNA (lncRNA WT1-AS) and microRNA-186-5p (miR-186-5p). Reverse transcription-quantitative PCR analysis was performed to determine lncRNA WT1-AS and miR-186-5p levels. An oxygen glucose deprivation (OGD)-induced SH-SY5Y cell injury model was established. Cell viability and apoptosis were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and flow cytometric assays, respectively. Caspase 3 activity was evaluated using a caspase 3 activity detection kit. The results showed that miR-186-5p is a direct target of the lncRNA WT1-AS. In addition, lncRNA WT1-AS levels were downregulated and miR-186-5p levels were upregulated in the blood samples of patients with ischemic stroke and OGD-induced SH-SY5Y cells. WT1-AS overexpression promoted OGD-induced cell viability and reduced the cell apoptosis and caspase 3 activity. However, these effects were reversed by miR-186-5p overexpression. Furthermore, the results demonstrated that the X-linked inhibitor of apoptosis (XIAP) was directly targeted by miR-186-5p. Similarly, transfection with the miR-186-5p inhibitor reduced OGD-induced neuronal damage by upregulating XIAP expression. In conclusion, lncRNA WT1-AS attenuates hypoxia/ischemia-induced neuronal injury in cerebral ischemic stroke through the miR-186-5p/XIAP axis.

Keywords: X-linked inhibitor of apoptosis; cerebral ischemic stroke; microRNA-186-5p; oxygen glucose deprivation; wilms tumor 1 antisense RNA.

Figure 1

Expression levels of lncRNA WT1-AS and miR-186-5p in patients with ischemic stroke. (a) Interaction between miR-186-5p and WT1-AS 3′-UTR was predicted using the Starbase prediction software. (b) Dual luciferase reporter gene assay was used to verify the interaction between WT1-AS and miR-186-5p in 293T cells co-transfected with miR-186-5p mimics and WT or MUT WT1-AS 3′-UTR reporter plasmids. (c and d) Expression levels of WT1-AS and miR-186-5p in blood samples of patients with ischemic stroke and healthy individuals. ^** P < 0.01 vs control mimics group; ^## P < 0.01 vs healthy control group. GraphPad Prism 6.0 software (GraphPad Software, Inc.) was used for creation of the figure.

Figure 2

Expression levels of lncRNA WT1-AS/miR-186-5p in OGD-induced SH-SY5Y cells. (a) RT-qPCR analysis results showing lncRNA WT1-AS expression levels. (b) RT-qPCR analysis results showing miR-186-5p expression levels. ^** P < 0.01 vs control group. GraphPad Prism 6.0 software (GraphPad Software, Inc.) was used for creation of the figure.

Figure 3

lncRNA WT1-AS negatively regulates miR-186-5p expression levels in SH-SY5Y cells. (a) RT-qPCR analysis results showing WT1-AS expression levels in SH-SY5Y cells transfected with the control or WT1-AS-plasmids. (b) RT-qPCR analysis results showing miR-186-5p expression levels in SH-SY5Y cells transfected with the control or miR-186-5p mimics. (c) RT-qPCR analysis showing the expression levels of WT1-AS in SH-SY5Y cells transfected with the control plasmid, miR-186-5p mimics, WT1-AS plasmid + control mimics, or WT1-AS plasmid + miR-186-5p mimics. (d) RT-qPCR analysis showing the expression levels of miR-186-5p in SH-SY5Y cells transfected with the control plasmid, miR-186-5p mimics, WT1-AS plasmid + control mimics, or WT1-AS plasmid + miR-186-5p mimics. ^** P < 0.01 vs control plasmid group; ^## P < 0.01 vs control mimics group; ^&& P < 0.01 vs WT1-AS plasmid + control mimics group. GraphPad Prism 6.0 software (GraphPad Software, Inc.) was used for creation of the figure.

Figure 4

Effect of lncRNA WT1-AS on OGD-induced nerve cell injury. (a) RT-qPCR analysis results showing microRNA-186-5p expression levels. (b) RT-qPCR analysis results showing lncRNA WT1-AS expression levels. (c) A MTT assay was used to assess the cell viability. (d) A flow cytometric assay was used to evaluate cell apoptosis. (e) The apoptosis ratio is presented. (f) Caspase 3 activity was detected using the corresponding kit. ^** P < 0.01 vs control group; ^## P < 0.01 vs OGD + control mimics group; ^&& P < 0.01 vs OGD + WT1-AS plasmid + control mimics group. GraphPad Prism 6.0 software (GraphPad Software, Inc.) was used for creation of the figure.

Figure 5

XIAP is directly targeted by miR-186-5p. (a) The interaction between miR-186-5p and XIAP 3′-UTR was predicted by TargetScan. (b) A dual luciferase reporter gene assay was performed to verify the interaction between miR-186-5p and XIAP in 293T cells co-transfected with miR-186-5p mimics and WT or MUT XIAP 3′-UTR reporter plasmids. (c) Expression levels of XIAP in blood samples of patients with ischemic stroke and healthy individuals.^** P < 0.01 vs control mimics group; ^## P < 0.01 vs Healthy control group. GraphPad Prism 6.0 software (GraphPad Software, Inc.) was used for creation of the figure.

Figure 6

miR-186-5p negatively regulates XIAP expression levels in SH-SY5Y cells. (a) RT-qPCR assay results showing miR-186-5p expression levels in SH-SY5Y cells transfected with the inhibitor control or miR-186-5p inhibitors. (b) RT-qPCR assay results showing XIAP expression levels in SH-SY5Y cells transfected with the control or XIAP siRNAs. (c) RT-qPCR assay results showing XIAP expression levels in SH-SY5Y cells transfected with the miR-186-5p inhibitor + control siRNA or miR-186-5p inhibitor + XIAP siRNA. (d) Western blotting analysis results showing the protein expression levels of XIAP. (e) XIAP/GAPDH. ^** P < 0.01 vs control inhibitor group; ^## P < 0.01 vs control siRNA group; ^&& P < 0.01 vs miR-186-5p inhibitor + control siRNA group. GraphPad Prism 6.0 software (GraphPad Software, Inc.) was used for creation of the figure.

Figure 7

miR-186-5p inhibitor attenuates OGD-induced neuronal injury by upregulating XIAP expression. (a) RT-qPCR analysis results showing miR-186-5p expression levels. (b) RT-qPCR analysis results showing XIAP expression levels. (c) MTT assay was used to assess the cell viability. (d) Flow cytometric assay was carried out to evaluate cell apoptosis. (e) The cell apoptosis ratio is presented. (f) Caspase 3 activity was detected using the corresponding kit. ^** P < 0.01 vs control group; ^## P < 0.01 vs OGD + inhibitor control group; ^&& P < 0.01 vs OGD + miR-186-5p inhibitor + control siRNA group. GraphPad Prism 6.0 software (GraphPad Software, Inc.) was used for creation of the figure.