MicroRNA-200b-3p promotes endothelial cell apoptosis by targeting HDAC4 in atherosclerosis

Fig. 1. The up-regulation of miR-200 family members in EAT from CAD patients. a The Volcano plot of miRNA expression fold change between CAD and CON (x axis, log2 fold change; y axis, p value significance). Up-regulated genes (Log2 FC > 1) in CAD samples are indicated by a red dot, and Down-regulated genes (Log2 FC < − 1) in CAD samples are indicated by a green dot. b Heat-map of the 250 differentially expressed transcripts. Red color indicated higher expression level; green color indicated lower expression level. c–g The expression levels of hsa-miR-200a-3p (c), hsa-miR-200b-3p (D), hsa-miR-200c-3p (e), hsa-miR-141-3p (f) and hsa-miR-429 (g) in EAT from CAD patients (n = 16) and CON patients (n = 5) were accessed by qRT-PCR, and normalized with U6. Unpaired t test was used for comparison between groups

Fig. 2. Oxidative stress response promotes the expression of miR-200 family in endothelial cells. a–d The expression levels of hsa-miR-200b-3p (a), hsa-miR-200c-3p (b), hsa-miR-141-3p (c) and hsa-miR-429 (d) in HUVECs with H2O2 (100 μM) and NAC (10 μM) were accessed by qRT-PCR, and normalized with U6. e HUVECs were stimulated with LPS (1 μg/ml) for 12 h, the expression levels of hsa-miR-200b-3p, hsa-miR-200c-3p, hsa-miR-141-3p and hsa-miR-429 were accessed by qRT-PCR, and normalized with U6. Unpaired t test was used for comparison between groups, *p < 0.05, **p < 0.01

Fig. 3. The function prediction of miR-200 family. a The targets genes of miR-200 family members, which was predicted by TargetScan database. Then the intersection targets of two sub-group were analyzed. b The top 5 enriched KEGG pathway of the intersection targets were analyzed by DAVID database

Fig. 4. Overexpression of hsa-miR-200b-3p exacerbates cell apoptosis induced by H2O2 and ox-LDL. a HUVECs were transfected with negative control (NC) mimic (50 nM) and hsa-miR-200b-3p (miR-200b) mimic (50 nM) for 24 h, then qRT-PCR for hsa-miR-200b-3p was used to access the transfection efficiency (n = 4 for each group). b HUVECs were transfected with NC mimic and miR-200b mimic for 24 h, then cells were stimulated with H2O2 (50 μM) or ox-LDL (100 μg/ml) for 6 h. The apoptosis of HUVECs was detected by Annexin V and PI staining. c The apoptosis ratio of HUVECs was calculated (n = 3 for each group). d The mRNA levels of anti-apoptosis gene BCL2 and pro-apoptosis gene BAX in H2O2 stimulated HUVECs were examined by qRT-PCR (n = 4 for each group). e The mRNA levels of BCL2 in ox-LDL stimulated HUVECs were examined by qRT-PCR (n = 4 for each group). f The protein levels of BCL2 and BAX in ox-LDL stimulated HUVECs were examined by western blot, and normalized with GAPDH. The right histogram was the ratio of BCL2 and BAX (n = 3 for each group). Full-length blots/gels are presented in Additional files 2 and 3: Figure S1 and S2. g The activity of Caspase3/7 in H2O2 stimulated HUVECs was examined…

Fig. 5. HDAC4 is the target gene of miRNA-200b-3p. a Identification the binding sites of hsa-miR-200b-3p in the HDAC4 3′-UTR (Red). b HUVECs were transfect with NC mimic and miR-200b mimic for 24 h, the mRNA level of HDAC4 in HUVECs were examined by qRT-PCR, and normalized with GAPDH. (n = 4 for each group). c The protein level HDAC4 in HUVECs was examined by western blot, and normalized with β-Actin (n = 3 for each group). Full-length blots/gels are presented in Additional file 4: Figure S3. d HUVECs were transfect with NC/miR-200b mimic with control adenovirus (Con-Ad) or HDAC4 over-expression adenovirus (HDAC4-Ad) for 48 h, the expression of HDAC4 was accessed by qRT-PCR (n = 4 for each group). e Con-Ad or HDAC4-Ad transfected HUVECs were stimulated with ox-LDL for 6 h. The apoptosis rate of HUVECs was accessed by Annexin V and PI staining (n = 4 for each group). Unpaired t test was used for comparison between groups, *p < 0.05

Fig. 6. The illustration of the explained mechanisms for miR-200b-3p in endothelial apoptosis