Fig 1: Effect of miR-140-5p knockdown and ox-LDL treatment on VSMC viability, migration, invasion and apoptosis. (A) VSMCs were identified via immunofluorescence (magnification, ×200). (B) Transfection efficiency of miR-140-5p inhibitor. (C) miR-140-5p and ROBO4 expression levels in miR-140-5p inhibitor-transfected and ox-LDL-treated VMSCs. (D) ROBO4 protein expression levels in miR-140-5p inhibitor-transfected and ox-LDL-treated human VSMCs (E) Bcl-2 and MMP2 expression levels were measured by reverse transcription-quantitative PCR. Human VSMC (F) viability, (G) migration, invasion (magnification, ×200) and (H) apoptosis following miR-140-5p inhibitor transfection and ox-LDL treatment. *P<0.05, **P<0.01, ***P<0.001 vs. NC or control; #P<0.05, ##P<0.01 vs. ox-LDL + NC. miR, microRNA; ox-LDL, oxidized-low density lipoprotein; VSMC, vascular smooth muscle cell; ROBO4, roundabout guidance receptor 4; MMP2, matrix metallopeptidase 2; α-SMA, α-smooth muscle actin; NC, negative control; OD, optical density.
Fig 2: miR-140-5p mediates VSMC apoptosis, migration and invasion by targeting ROBO4. (A) Transfection efficiency of ROBO4 siRNA. (B) Effects of ox-LDL, miR-140-5p inhibitor and ROBO4 siRNA on ROBO4 expression levels in VSMCs. (C) Bcl-2 and MMP2 expression levels were measured by reverse transcription-quantitative PCR. Effect of ox-LDL, miR-140-5p inhibitor and ROBO4 siRNA on VSMC (D) apoptosis, (E) migration and invasion (magnification, ×200). *P<0.05, **P<0.01, ***P<0.001 vs. NC or control; #P<0.05 vs. ox-LDL + NC; $P<0.05 vs. ox-LDL + inhibitor + siRNA NC. miR, microRNA; VSMC, vascular smooth muscle cell; ROBO4, roundabout guidance receptor 4; siRNA, small interfering RNA; ox-LDL, oxidized-low density lipoprotein; MMP2, matrix metallopeptidase 2; NC, negative control.
Fig 3: Investigation of miR-140-5p binding to ROBO4 mRNA in VSMCs. (A) The predicted binding site between miR-140-5p and the 3′-UTR sequences of ROBO4 mRNA. (B) Verification of miR-140-5p binding to the 3′-UTR sequences of ROBO4 mRNA in human VSMCs. (C) Expression of ROBO4 in VSMCs following transfection with miR-140 mimic. **P<0.01, vs. NC. miR, microRNA; ROBO4, roundabout guidance receptor 4; VSMC, vascular smooth muscle cell; 3′-UTR, 3′-untranslated region; WT, wild-type; MUT, mutant; NC, negative control; ox-LDL, oxidized-low density lipoprotein.
Fig 4: Increase in miR-140-5p expression and decrease in ROBO4 expression associated with AS pathogenesis. (A) miR-140-5p expression levels in healthy artery tissues and artery tissues obtained from patients with AS. **P<0.01 vs. normal. (B) ROBO4 protein expression levels in ox-LDL-treated human VSMCs. (C) miR-140-5p and ROBO4 gene expression levels in ox-LDL-treated human VSMCs. *P<0.05, **P<0.01 vs. blank group. miR, microRNA; ROBO4, roundabout guidance receptor 4; AS, atherosclerosis; ox-LDL, oxidized-low density lipoprotein; VMSC, vascular smooth muscle cell.
Fig 5: Effects of ROBO4 overexpression and ox-LDL treatment on VSMC viability, migration, invasion and apoptosis. (A) Transfection efficiency of ROBO4 overexpression. ROBO4 (B) mRNA and (C) protein expression levels in human VSMCs following ROBO4 overexpression and ox-LDL treatment. (D) Bcl-2 and MMP2 expression levels were measured by reverse transcription-quantitative PCR. Effect of ROBO4 overexpression and ox-LDL treatment on human VSMC (E) viability, (F) migration, invasion (magnification, ×200) and (G) apoptosis. *P<0.05, **P<0.01, ***P<0.001 vs. NC or control; #P<0.05, ##P<0.01 vs. ox-LDL + NC. ROBO4, roundabout guidance receptor 4; ox-LDL, oxidized-low density lipoprotein; VSMC, vascular smooth muscle cell; MMP2, matrix metallopeptidase 2; NC, negative control; OD, optical density.
Supplier Page from Abcam for Anti-Robo4 antibody