Long noncoding RNA MALAT1 inhibits apoptosis induced by oxygen-glucose deprivation and reoxygenation in human brain microvascular endothelial cells

Figures

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  Figure 1.. Expression of MALAT1 and apoptosis in brain vascular endothelial cells under OGD-R. Human brain microvascular endothelial cells were cultured under OGD-R. (A) The expression levels of MALAT1 were measured at 6, 9, 12, 24 and 36 h post-reoxygenation using reverse transcription-quantitative polymerase chain reaction and expressed as fold changes to that of the control cells under normal culture conditions (designated as 1); each group was normalized against RPLP0 mRNA expression levels as a reference gene. (B) The apoptotic rate of cells under OGD-R was measured at 6, 9, 12, 24 and 36 h post-reoxygenation with a microplate reader-based apoptosis detection kit. Cells under normal culture conditions were used as a control. Each experiment was performed in duplicate and repeated three times. *P<0.05 vs. normal; #P<0.05 vs. 6 h; ^P<0.05 vs. 9 h; &P<0.05 vs. 12 h. MALAT1, metastasis-associated lung adenoma transcript 1; OGD-R, oxygen-glucose deprivation and reoxygenation.

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  Figure 2.. Overexpression and knockdown of MALAT1 in brain vascular endothelial cells in the presence or absence of OGD-R. Human brain microvascular endothelial cells were transduced with human LV-MALAT1 or human MALAT1-siRNA to overexpress or knockdown MALAT1, respectively. Cells transduced with VC or SC were used as controls. Cells transduced with human LV-MALAT1 were also treated with a phosphatidylinositol 3-kinase inhibitor, Wortmannin (50 µM), during reoxygenation. The expression level of MALAT1 (A) in the absence or (B) presence of OGD-R (specifically, at 6, 9, 12, 24 and 36 h post-reoxygenation) was measured via reverse transcription-quantitative polymerase chain reaction and expressed as fold changes to that of the control cells under normal culture conditions (designated as 1); each group was normalized against RPLP0 mRNA expression levels as a reference gene. Each experiment was performed in duplicate and repeated three times. *P<0.05 vs. SC and VC; #P<0.05 vs. 6 h; ^P<0.05 vs. 9 h; &P<0.05 vs. 12 h. MALAT1, metastasis-associated lung adenoma transcript 1; OGD-R, oxygen-glucose deprivation and reoxygenation; LV-MALAT1, MALAT1 lentivirus; MALAT1-siRNA, MALAT1 siRNA lentivirus; VC,…

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  Figure 3.. Apoptosis in brain vascular endothelial cells with overexpression or knockdown of MALAT1 in the presence or absence of OGD-R. Human brain microvascular endothelial cells were transduced with human LV-MALAT1 or MALAT1-siRNA to overexpress or knockdown MALAT1, respectively. Cells transduced with VC or SC were used as controls. Cells transduced with human MALAT1 lentivirus were also treated with phosphatidylinositol 3-kinase inhibitor, Wortmannin (50 µM), during reoxygenation. The apoptotic rate of cells (A) in the absence or (B) the presence of OGD-R (specifically, at 6, 9, 12, 24 and 36 h post-reoxygenation) was measured with a microplate reader-based apoptosis detection kit. Each experiment was performed in duplicate and repeated three times. *P<0.05 vs. SC and VC; #P<0.05 vs. 6 h; ^P<0.05 vs. 9 h; &P<0.05 vs. 12 h. MALAT1, metastasis-associated lung adenoma transcript 1; OGD-R, oxygen-glucose deprivation and reoxygenation; LV-MALAT1, MALAT1 lentivirus; MALAT1-siRNA, MALAT1 siRNA lentivirus; VC, blank control lentivirus; SC, scrambled siRNA lentivirus; LV-MALAT1+WN, MALAT1 lentivirus-Wortmannin.

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  Figure 4.. TUNEL staining in brain vascular endothelial cells with overexpression or knockdown of MALAT1 in the presence or absence of OGD-R. Human brain microvascular endothelial cells were transduced with human LV-MALAT1 or MALAT1-siRNA to overexpress or knockdown MALAT1, respectively. Cells transduced with VC or SC lentivirus were used as controls. Cells transduced with human LV-MALAT1 were also treated with phosphatidylinositol 3-kinase inhibitor, Wortmannin (50 µM), during reoxygenation. The apoptotic rate of cells (A) in the absence or (B) presence of OGD-R at 36 h post-reoxygenation was measured with a TUNEL staining kit. The images present cell nuclei stained by DAPI (blue) and DNA fragmentation (green). (C) The TUNEL-positive/apoptosis rate is shown as the percentage of total cells in histograms. Each experiment was repeated three times.*P<0.05 vs. SC and VC. TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; MALAT1, metastasis-associated lung adenoma transcript 1; OGD-R, oxygen-glucose deprivation and reoxygenation; LV-MALAT1, MALAT1 lentivirus; MALAT1-siRNA, MALAT1 siRNA lentivirus; VC, blank control lentivirus; SC, scrambled siRNA lentivirus; LV-MALAT1+WN,…

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  Figure 5.. Caspase 3 activity in brain vascular endothelial cells with overexpression or knockdown of MALAT1 in the presence or absence of OGD-R. Human brain microvascular endothelial cells were transduced with human LV-MALAT1 MALAT1 or MALAT1-siRNA to overexpress or knockdown MALAT1, respectively. Cells transduced with VC or SC lentivirus were used as controls. Cells transduced with human LV-MALAT1 were also treated with phosphatidylinositol 3-kinase inhibitor, Wortmannin (50 µM), during reoxygenation. The caspase 3 activity in cells (A) in the absence or (B) presence of OGD-R (specifically, at 6, 9, 12, 24 and 36 h post-reoxygenation) was measured with a colorimetric caspase 3 assay kit and and expressed as fold changes to that of cells under normal culture conditions (designated as 1). Each experiment was performed in duplicate and repeated three times. *P<0.05 vs. SC and VC; #P<0.05 vs. 6 h; ^P<0.05 vs. 9 h; &P<0.05 vs. 12 h. MALAT1, metastasis-associated lung adenoma transcript 1; OGD-R, oxygen-glucose deprivation and reoxygenation; LV-MALAT1, MALAT1 lentivirus; MALAT1-siRNA, MALAT1 siRNA lentivirus; VC, blank control lentivirus; SC, scrambled siRNA lentivirus; LV-MALAT1+WN, MALAT1…

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  Figure 6.. ROS production in brain vascular endothelial cells with overexpression or knockdown of MALAT1 in the presence or absence of OGD-R. Human brain microvascular endothelial cells were transduced with human LV-MALAT1 or MALAT1-siRNA to overexpress or knockdown MALAT1, respectively. Cells transduced with VC or SC lentivirus were used as controls. Cells transduced with human LV-MALAT1 were also treated with phosphatidylinositol 3-kinase inhibitor, Wortmannin (50 µM), during reoxygenation. ROS production in cells (A) in the absence or (B) presence of OGD-R (specifically, at 6, 9, 12, 24 and 36 h post-reoxygenation) was measured with a fluorescence cellular ROS detection assay kit and expressed as fold changes to that of the control cells under normal culture conditions (designated as 1). Each experiment was performed in duplicate and repeated three times. *P<0.05 vs. SC and VC; #P<0.05 vs. 6 h; ^P<0.05 vs. 9 h; &P<0.05 vs. 12 h. ROS, reactive oxygen species; MALAT1, metastasis-associated lung adenoma transcript 1; OGD-R, oxygen-glucose deprivation and reoxygenation; LV-MALAT1, MALAT1 lentivirus; MALAT1-siRNA, MALAT1 siRNA lentivirus; VC, blank control lentivirus; SC, scrambled siRNA…

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  Figure 7.. PI3K activity in brain vascular endothelial cells with overexpression or knockdown of MALAT1 in the presence or absence of OGD-R. Human brain microvascular endothelial cells were transduced with human LV-MALAT1) or MALAT1-siRNA to overexpress or knockdown MALAT1, respectively. Cells transduced with VC or SC were used as controls. Cells transduced with human LV-MALAT1 were also treated with PI3K inhibitor, Wortmannin (50 µM), during reoxygenation. The PI3K activity in cells (A) in the absence or (B) the presence of OGD-R (specifically, at 6, 9, 12, 24 and 36 h post-reoxygenation) was measured with a PI3K activity ELISA kit and expressed as fold changes to that of the control cells under normal culture conditions (designated as 1). Each experiment was performed in duplicate and repeated three times. *P<0.05 vs. SC and VC; #P<0.05 vs. 6 h; ^P<0.05 vs. 9 h; &P<0.05 vs. 12 h. PI3K, phosphatidylinositol 3-kinase; MALAT1, metastasis-associated lung adenoma transcript 1; OGD-R, oxygen-glucose deprivation and reoxygenation; LV-MALAT1, MALAT1 lentivirus; MALAT1-siRNA, MALAT1 siRNA lentivirus; VC, blank control lentivirus; SC, scrambled siRNA lentivirus; LV-MALAT1+WN, MALAT1…

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  Figure 8.. p-Akt levels in brain vascular endothelial cells with overexpression or knockdown of MALAT1 in the presence or absence of OGD-R. Human brain microvascular endothelial cells were transduced with human MALAT1 LV-MALAT1 or MALAT1-siRNA to overexpress or knockdown MALAT1, respectively. Cells transduced with VC or SC were used as controls. Cells transduced with human LV-MALAT1 were also treated with phosphatidylinositol 3-kinase inhibitor, Wortmannin (50 µM), during reoxygenation. Levels of total Akt and p-Akt at ser473 were determined by western blot analyses (A) in the absence or (B) the presence of OGD-R (specifically, at 6, 9, 12, 24 and 36 h post-reoxygenation). Lanes 1, 6 and 11: NC; lanes 2, 7 and 12: VC; lanes 3, 8 and 13: SC; lanes 4, 9 and 14: LV-MALAT1; lanes 5, 10 and 15: MALAT1-siRNA. Lanes 1–5: 6 h post-reoxygenation; lanes 6–10: 9 h post-reoxygenation; lanes 11–15: 36 h post-reoxygenation. The total Akt level was not significantly altered by OGD-R. Density of the p-Akt (ser473) blot was normalized against that of total Akt to obtain a relative p-Akt blot density. Each experiment was repeated three times. *P<0.05 vs. NC, SC and VC; #P<0.05 vs. 6 h. p-Akt, phosphorylated…