Effect of miR-655-3p targeting FOXC1 and CXCR4 on angiogenesis in hypoxic human umbilical vein endothelial cells

Objective  To explore the effect and mechanism of microRNA-655-3p (miR-655-3p) targeting forkhead box protein C1 (FOXC1) and chemokine receptor 4 (CXCR4) on human umbilical vein endothelial cells (HUVECs).

Methods  HUVECs in the logarithmic growth phase were divided into the normoxia control group and hypoxia groups treated for 6, 12, 24 and 48 hours, respectively. The expression of miR-655-3p, FOXC1 and CXCR4 mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR), and the expression of FOXC1 and CXCR4 proteins was determined by Western blot. In addition, HUVECs in logarithmic phase were assigned into five groups: 24-hour hypoxia control group, miR-655-3p mimics control group, miR-655-3p mimics group, miR-655-3p inhibitor control group, and miR-655-3p inhibitor group. Among them, the 24-hour hypoxia control group was only treated with hypoxia, and the other groups were transfected with corresponding lentiviruses on the basis of hypoxia treatment. The expression of miR-655-3p was detected by RT-qPCR. The expression of FOXC1 and CXCR4 proteins was measured by Western blot. Cell apoptosis, proliferation, migration and tube formation ability were evaluated by flow cytometry, EdU cell proliferation assay, Transwell assay and Matrigel tube formation assay, respectively. A dual-luciferase reporter assay was adopted to verify the targeting relationship between miR-655-3p and FOXC1/CXCR4.

Results  The expression of miR-655-3p was the lowest and the relative expression of CXCR4 mRNA was the highest in the 24-hour hypoxia group. The relative expression of FOXC1 mRNA was the highest in the 12-hour hypoxia group. The relative expression of FOXC1 protein was the highest in the 24-hour hypoxia group, and the relative expression of CXCR4 protein was the highest in the 48-hour hypoxia group. The relative expression of miR-655-3p in the miR-655-3p mimics group was significantly higher than that in the 24-hour hypoxia control group, miR-655-3p mimics control group, miR-655-3p inhibitor control group and miR-655-3p inhibitor group, showing statistically significant differences (all P<0.001). The relative expression of FOXC1 and CXCR4 proteins, cell proliferation rate, the number of migrating cells, and the number of tubes formed in the miR-655-3p mimics group were significantly lower than those in the 24-hour hypoxia control group, miR-655-3p mimics control group, and miR-655-3p inhibitor control group, and were significantly lower than those in the miR-655-3p inhibitor group (all P<0.05). The cell apoptosis rates in the 24-hour hypoxia control group, miR-655-3p mimics control group, miR-655-3p mimics group, miR-655-3p inhibitor control group, and miR-655-3p inhibitor group were (20.44±1.07)%, (23.54±1.98)%, (32.91±1.33)%, (19.86±0.51)% and (11.25±0.46)%, respectively, with a significant overall difference ( F=124.80, P<0.001). The cell apoptosis rate in the miR-655-3p mimics group was higher than that in the miR-655-3p mimics control group, and the apoptosis rate in the miR-655-3p inhibitor group was lower than that in the miR-655-3p inhibitor control group, showing statistically significant differences (both P<0.05). Bioinformatics software predicted that the miR-655-3p continuous binding site targeting sequence 5′-UGUAUUA-3′ in the 3′untranslated region of FOXC1 and CXCR4. The luciferase activities of wild-type FOXC1 and wild-type CXCR4 in the miR-655-3p mimics group were significantly lower than those in the miR-655-3p mimics control group ( t=5.37, 4.05; both P<0.05).

Conclusions  Overexpression of miR-655-3p may enhance cell apoptosis and inhibit cell proliferation, migration and tube formation by negatively regulating FOXC1/CXCR4.