MicroRNA‑93 regulates angiogenesis in peripheral arterial disease by targeting CDKN1A

Abstract

MicroRNAs (miRNAs) are considered to be critical mediators of gene expression with respect to tumor progression, although their role in ischemia‑induced angiogenesis is poorly characterized, including in peripheral arterial disease (PAD). Furthermore, the underlying mechanism of action of specific miRNAs in PAD remains unknown. Reverse transcription‑quantitative polymerase chain reaction analysis revealed that microRNA‑93 (miR‑93) was significantly upregulated in patients with PAD and in the EA.hy926 endothelial cells in response to hypoxia. Additionally, miRNA (miR)‑93 promoted angiogenesis by enhancing proliferation, migration and tube formation. Cyclin dependent kinase inhibitor 1A (CDKN1A), verified as a potential target gene of miR‑93, was inhibited by overexpressed miR‑93 at the protein and mRNA expression levels. Furthermore, a hind‑limb ischemia model served to evaluate the role of miR‑93 in angiogenesis in vivo, and the results demonstrated that miR‑93 overexpression enhanced capillary density and perfusion recovery from hind‑limb ischemia. Taken together, miR‑93 was indicated to be a promising target for pharmacological regulation to promote angiogenesis, and the miR‑93/CDKN1A pathway may function as a novel therapeutic approach in PAD.

Figure 1.

Expression levels of miR-93 in patients with PAD. (A) RT-qPCR analysis of miR-93 expression in patients with PAD (n=48) and controls (n=25). **P<0.01 vs. control group. (B) RT-qPCR analysis of miR-93 expression in patients with differing degrees of PAD. *P<0.05 vs. Level 1. The experiment was repeated three times. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miR, microRNA; PAD, peripheral arterial disease.

Figure 2.

CDKN1A is a potential target of miR-93. (A) Schematic representation of the CDKN1A 3′-UTR as a direct target of miR-93. (B) Luciferase activity of EA.hy926 cells co-transfected with the WT or Mut CDKN1A 3′-UTR reporter genes or negative control miRNA mimics. **P<0.01 vs. pMIR-CDKN1A-Mut. (C) Quantification of CDKN1A mRNA expression levels by reverse transcription-quantitative polymerase chain reaction. *P<0.05 vs. NC. (D) CDKN1A protein expression was determined by western blotting in EA.hy926 cells co-transfected with miR-93 or control, and the quantification was performed using Image J. n=3. **P<0.01 vs. miR-NC. The experiment was repeated three times. CDKN1A, cyclin dependent kinase inhibitor 1A; miR, microRNA; UTR, untranslated region; NC, negative control; WT, wild-type; Mut, mutant.

Figure 3.

Effects of miR-93 in EA.hy926 cells in response to hypoxia. (A) RT-qPCR analysis of miR-93 expression in EA.hy926 cells when cultured in normal and hypoxic conditions. **P<0.01 vs. NC. (B) RT-qPCR analysis of miR-93 expression in EA.hy926 cells transfected with the miR-93 vector. **P<0.01 vs. NC. (C) The proliferation of cells transfected with miR-93 or the control when cultured in hypoxic conditions was determined using an MTT assay. *P<0.05 vs. hypoxia. (D) Representative images of cell migration following transfection with miR-93 or the control when cultured in hypoxic conditions at 0 and 12 h. Scale bar, 50 µm. (E) Representative images of tube formation of cells transfected with miR-93 or the control when cultured in hypoxic conditions for 12 h. Scale bar, 50 µm. n=3. The experiment was repeated three times. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miR, microRNA; NC, negative control.

Figure 4.

Overexpression of miR-93 in the hind-limb ischemic model to improve perfusion recovery. (A) miR-93 significantly increased the blood flow ratio, and improved (B) tissue necrosis and (C) ambulatory impairment. **P<0.01 vs. NC. (D) Representative images of H&E and CD31 staining. Scale bar, 100 µm. n=6. The experiment was repeated three times. miR, microRNA; H&E, hematoxylin and eosin; CD31, platelet endothelial cell adhesion molecule; NC, negative control.