Circular RNA_0033596 aggravates endothelial cell injury induced by oxidized low-density lipoprotein via microRNA-217-5p /chloride intracellular channel 4 axis

Figures & data

Figure 1. Circ_0033596 is up-regulated in HUVECs treated with ox-LDL.

(a) The generation process of circ_0033596 was queried from circBase database, and a diagram was drawn. (b) HUVECs were treated with 100 μg/ml ox-LDL, and qRT-PCR was conducted to detect the expression of circ_0033596 in the treated cells and untreated cells.***P < 0.001.

Figure 2. Effects of circ_0033596 overexpression or knockdown on HUVEC viability, cell cycle progression and apoptosis.

(a) Circ_0033596 overexpression plasmid or si-circ_0033596#1/2 was transfected into ox-LDL-induced HUVECs, and qRT-PCR was employed to detect circ_0033596 expression to verify the transfection efficiency. (b) HUVEC viability was detected by CCK-8 assay after the transfection. (c) The cell cycle progression of HUVECs was detected via flow cytometry after the transfection. (d) Flow cytometry was utilized to detect HUVEC apoptosis after the transfection.*P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3. Targeted relationship between circ_0033596 and miR-217-5p.

(a) The StarBase database was adopted to predict the binding site between circ_0033596 and miR-217-5p. (b) Dual-luciferase reporter gene assay was conducted to verify the effects of miR-217-5p overexpression on the luciferase activity of circ_0033596 WT and circ_0033596 MUT. (c) RIP assay was performed to further verify the binding relationship between circ_0033596 and miR-217-5p. (d) qRT-PCR was used to detect the effect of circ_0033596 overexpression or knockdown on miR-217-5p expression in HUVECs.***P < 0.001.

Figure 4. Circ_0033596 modulates the viability, cell cycle progression and apoptosis of HUVECs via targeting miR-217-5p.

(a) Ox-LDL-induced HUVECs were transfected with circ_0033596 overexpression plasmids and miR-217-5p mimics, and qRT-PCR was utilized to detect miR-217-5p expression to verify the transfection efficiency. (b) Circ_0033596 overexpression plasmids and miR-217-5p mimics were transfected into ox-LDL-induced HUVECs, and the cell viability was detected using CCK-8 assay. (c) The cell cycle progression of HUVECs was detected through flow cytometry after the transfection. (d) Flow cytometry was employed to detect the apoptosis of HUVECs after the transfection.*P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 5. Circ_0033596 up-regulates CLIC4 expression by adsorbing miR-217-5p.

(a) The StarBase and TargetScan databases were utilized to predict the downstream target genes of miR-217-5p, and the Venn diagram was applied to display the number of target genes. (b) The KEGG database was adopted to perform a pathway enrichment analysis of the target genes of miR-217-5p. (c) Binding sites of miR-217-5p with CLIC4 WT and CLIC4 MUT luciferase reporters. (d) Dual-luciferase reporter gene assay was used to detect the effects of miR-217-5p overexpression on the luciferase activity of CLIC4 WT and CLIC4 MUT. (e) qRT-PCR and Western blot were conducted to detect the effects of transfection with miR-217-5p mimics or inhibitors on CLIC4 mRNA and protein expression in HUVECs. (f) qRT-PCR and Western blot were utilized to detect the effects of circ_0033596 and miR-217-5p on CLIC4 mRNA and protein expression in HUVECs.***P < 0.001.