miRNA‑504 inhibits p53‑dependent vascular smooth muscle cell apoptosis and may prevent aneurysm formation

Abstract

Abdominal aortic aneurysm (AAA) is a common disease that is associated with the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). VSMCs are regulated by microRNAs (miRNA). The aim of the present study was to identify miRNA sequences that regulate aortic SMCs during AAA. miRNA‑504 was identified using a miRNA PCR array and by reverse transcription‑quantitative polymerase chain reaction analysis, and its expression levels were observed to be downregulated in the aortic cells derived from patients with AAA when compared with controls. Transfection of SMCs with pMSCV‑miRNA‑504 vector was performed, and cell proliferation and the expression levels of proliferating cell nuclear antigen (PCNA), replication factor C subunit 4 (RFC4), B‑cell lymphoma‑2 (Bcl‑2) and caspase‑3/9 were measured by western blotting. The mechanisms underlying the effects of miRNA‑504 was then analyzed. The results demonstrated that overexpression of miRNA‑504 significantly upregulated the expression levels of PCNA, RFC4 and Bcl‑2, while caspase‑3/9 expression was significantly inhibited when compared with non‑targeting controls. In addition, miRNA‑504 overexpression was observed to promote the proliferation of SMCs. The expression level of the tumor suppressor, p53, which is known to be a direct target of miRNA‑504, was inhibited following transfection of SMCs with pMSCV‑miRNA‑504. In addition, the expression of the downstream targets of p53, p21 and Bcl‑like protein‑4, were significantly reduced following overexpression of miRNA‑504. These results revealed the anti‑apoptotic role of miRNA‑504 in SMCs derived from patients with AAA via direct targeting of p53.

Figure 1.

Identification and verification of the differential expression patterns of miRNAs in the aortic SMCs of patients with AAA. (A) Screening of miRNA sequences in serum samples from patients with AAA and paired controls using a PCR microarray. (B) Validation of microarray results for selected miRNAs that were significantly upregulated and downregulated in AAA samples when compared with controls, as determined by RT-qPCR analysis. To verify the accuracy of the microarray-based miRNA quantification, the six above-mentioned miRNAs (Let-7c, miR-499a, miR-144-3p, miR-192-5p, miR-504 and miR-542-3p) were re-examined using RT-qPCR. The results confirmed that the microarray data was reliable. (C) Confirmation of miRNA-504 expression in aortic SMCs derived from patients with AAA and paired controls by RT-qPCR analysis. **P<0.01 vs. controls. miRNA, microRNA; AAA, abdominal aortic aneurysm; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 2.

Effect of miRNA-504 on the proliferation of vascular SMCs. (A) The expression of miRNA-504, (B) the viability of aortic SMCs and (C) the protein expression levels of PCNA and RFC4 following transfection of aortic SMCs with pMSCV-miRNA-504 or pMSCV-NC vectors. (D) The expression of miRNA-504, (E) the viability of aortic SMCs and (F) the protein expression levels of PCNA and RFC4 following transfection of aortic SMCs with the miRNA-504 inhibitor or control. *P<0.05 and **P<0.01 vs. pMSCV-NC; ^#P<0.05 and ^##P<0.01 vs. the control. miRNA, microRNA; SMCs, smooth muscle cells; NC, negative control; PCNA, proliferating cell nuclear antigen; RFC4, replication factor C subunit 4.

Figure 3.

Effect of miRNA-504 expression on the apoptosis of vascular SMCs. (A) Decreased levels of apoptosis were observed in SMCs transfected with pMSCV-miRNA-504. (B) The expression of apoptosis-associated proteins as determined by western blot analysis. (C) Increased levels of apoptosis were observed in SMCs transfected with an miRNA-504 inhibitor. (D) The expression of apoptosis-associated proteins following inhibition of miRNA-504. **P<0.01 vs. pMSCV-NC; ^##P<0.01 vs. the control. miRNA, microRNA; SMCs, smooth muscle cells; Bcl-2, B-cell lymphoma-2; PI, propidium iodide.

Figure 4.

Validation of the direct interation between miRNA-504 and p53. (A) Bioinformatic analysis of the predicted interaction between miRNA-504 and p53. (B) The levels of luciferase activity following transfection of aortic SMCs with p53 3′-UTR-Mut and p53 3′-UTR-Wt sequences together with hsa-miRNA-504 (**P<0.01 vs. blank). (C) The levels of luciferase activity following transfection of aortic SMCs with p53 3′-UTR-Mut and p53 3′-UTR-Wt sequences together with miRNA-504 inhibitor (**P<0.01 vs. blank). (D) The protein expression levels of of p53, p21 and Bax, as determined by western blotting (**P<0.01 vs. control). miRNA, microRNA; SMCs, smooth muscle cells; Wt, wild-type; Mut, mutation; Bax, Bcl-2-like protein 4.