Figures & data
Figure 1. Decreased cell viability and temporarily increased lncRNA NORAD level after OGD/R induction. SH-SY5Y cells were cultured for 2, 4, 6, 8, 10, 12 h in glucose-free DMEM medium under hypoxic conditions (1% O2, 5% CO2, 94% N2). Subsequently, the cells were returned to normal DMEM medium and subjected to reoxygenation (21% O2, 5% CO2, 74% N2) continuously. (a) Cell viability was determined using CCK8 assay. (b) LncRNA NORAD level was detected by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001
Figure 2. Upregulation of lncRNA NORAD enhanced cell viability and suppressed cell apoptosis under OGD/R condition. OGD/R-injured SH-SY5Y cells were transfected with pcDNA3.1-NC or pcDNA3.1-NORAD. (a) The overexpression efficiency was validated by RT-qPCR analysis. (b) Cell viability was determined using CCK-8 assay. (c, d) Cell apoptosis was assessed using TUNEL assay. (e) Expression levels of Bcl-2, Bax and Cleaved caspase-3 were detected using western blot assay. **P < 0.01, ***P < 0.001
Figure 3. Downregulation of lncRNA NORAD reduced cell viability and promoted cell apoptosis under OGD/R condition. OGD/R-injured SH-SY5Y cells were transfected with sh-NC or sh-NORAD. (a) The silencing efficiency was validated by RT-qPCR analysis. (b) Cell viability was determined using CCK-8 assay. (c, d) Cell apoptosis was assessed using TUNEL assay. (e) Expression levels of Bcl-2, Bax and Cleaved caspase-3 were detected using western blot assay. *P < 0.05, **P < 0.01, ***P < 0.001
Figure 4. LncRNA NORAD exerted suppressive effects on oxidative stress and inflammation under OGD/R condition. OGD/R-injured SH-SY5Y cells were transfected with pcDNA3.1-NORAD or sh-NORAD. (a) The levels of ROS, MDA, LDH and SOD in cell suspension were determined by ROS Assay Kit, MDA Assay Kit, LDH Assay Kit and SOD Assay Kit. (b) The release of TNF-α, IL-1β and IL-6 was measured using ELISA Kits. *P < 0.05, **P < 0.01, ***P < 0.001
Figure 5. LncRNA NORAD served as a molecular sponge for miR-30a-5p and negatively regulated miR-30a-5p expression. (a) Bioinformatics analysis predicted the binding site of lncRNA NORAD to miR-30a-5p. (b) Cells were transfected with miR-30a-5p mimic and the overexpression efficiency was validated by RT-qPCR analysis. (c) Luciferase reporter assay verified the binding relationship of lncRNA NORAD and miR-30a-5p. (d) OGD/R-injured SH-SY5Y cells were transfected with sh-NC or sh-NORAD. miR-30a-5p level was detected by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001
Figure 6. Upregulation of lncRNA NORAD enhanced the viability and suppressed the apoptosis of OGD/R-injured SH-SY5Y cells by repressing miR-30a-5p expression. OGD/R-injured SH-SY5Y cells were transfected with pcDNA3.1-NORAD or co-transfected with pcDNA3.1-NORAD and miR-30a-5p mimic. (a) miR-30a-5p level was detected by RT-qPCR. (b) Cell viability was determined using CCK-8 assay. (c, d) Cell apoptosis was assessed using TUNEL assay. (e) Expression levels of Bcl-2, Bax and Cleaved caspase-3 were detected using western blot assay. *P < 0.05, **P < 0.01, ***P < 0.001
Figure 7. Upregulation of lncRNA NORAD alleviated oxidative stress and inflammation by repressing miR-30a-5p expression. OGD/R-injured SH-SY5Y cells were transfected with pcDNA3.1-NORAD or co-transfected with pcDNA3.1-NORAD and miR-30a-5p mimic. (a) The levels of ROS, MDA, LDH and SOD in cell suspension were determined by ROS Assay Kit, MDA Assay Kit, LDH Assay Kit and SOD Assay Kit. (b) The release of TNF-α, IL-1β and IL-6 was measured using ELISA Kits. **P < 0.01, ***P < 0.001
Figure 8. LncRNA NORAD regulated YWHAG expression by sponging miR-30a-5p. (a) Bioinformatics analysis predicted the binding site of miR-30a-5p to YWHAG. (b) Luciferase reporter assay verified the binding relationship of miR-30a-5p and YWHAG. (c) YWHAG level in OGD/R-injured SH-SY5Y cells was assessed by RT-qPCR. (d) Cells were transfected with miR-30a-5p mimic and YWHAG level was assessed by RT-qPCR. (e) Cells were transfected with pcDNA3.1-NORAD or co-transfected with pcDNA3.1-NORAD and miR-30a-5p mimic. YWHAG level was assessed by RT-qPCR. **P < 0.01, ***P < 0.001
Figure 9. Downregulation of lncRNA NORAD reduced the viability and promoted the apoptosis of OGD/R-injured SH-SY5Y cells by sponging miR-30a-5p to regulate YWHAG expression. OGD/R-injured SH-SY5Y cells were transfected with sh-NORAD or co-transfected with sh-NORAD and Ov-YWHAG. (a) Cells were transfected with Ov-YWHAG and RT-qPCR analysis was employed to check the overexpression efficiency. (b) Cell viability was determined using CCK-8 assay. (c, d) Cell apoptosis was assessed using TUNEL assay. (e) Expression levels of Bcl-2, Bax and Cleaved caspase-3 were detected using western blot assay. *P < 0.05, **P < 0.01, ***P < 0.001
Figure 10. Downregulation of lncRNA NORAD aggravated oxidative stress and inflammation by sponging miR-30a-5p to regulate YWHAG expression. OGD/R-injured SH-SY5Y cells were transfected with sh-NORAD or co-transfected with sh-NORAD and Ov-YWHAG. (a) The levels of ROS, MDA, LDH and SOD in cell suspension were determined by ROS Assay Kit, MDA Assay Kit, LDH Assay Kit and SOD Assay Kit. (b) The release of TNF-α, IL-1β and IL-6 was measured using ELISA Kits. *P < 0.05, **P < 0.01, ***P < 0.001
Data availability
The datasets analyzed during the current study are available from the corresponding author on reasonable request.