Yonsei Med J.  2019 Apr;60(4):352-359. 10.3349/ymj.2019.60.4.352.

Regulatory Mechanism of MicroRNA-145 in the Pathogenesis of Acute Aortic Dissection

• Li T
• Liu C
• Liu L
• Xia H
• Xiao Y
• Wang X
• Wang Y

• KMID: 2450190
• DOI: http://doi.org/10.3349/ymj.2019.60.4.352

Abstract

PURPOSE
Previous studies have confirmed that microRNAs play important roles in the pathogenesis of acute aortic dissection (AAD). Here, we aimed to explore the role of miR-145 and its regulatory mechanism in the pathogenesis of AAD.
MATERIALS AND METHODS
AAD tissue samples were harvested from patients with aortic dissection and normal donors. Rat aortic vascular smooth muscle cells (VSMCs) were transfected with miR-145 mimic/inhibitor or negative control mimic/inhibitor. Gene and protein expression was measured in human aortic dissection tissue specimens and VSMCs by qRT-PCR and Western blot. Luciferase reporter assay was applied to verify whether connective tissue growth factor (CTGF) was a direct target of miR-145 in VSMCs. Methyl thiazolyl tetrazolium assay was used to detect VSMC viability.
RESULTS
miR-145 expression was downregulated in aortic dissection tissues and was associated with the survival of patients with AAD. Overexpression of miR-145 promoted VSMC proliferation and inhibited cell apoptosis. Moreover, CTGF, which was increased in aortic dissection tissues, was decreased by miR-145 mimic and increased by miR-145 inhibitor. Furthermore, CTGF was confirmed as a target of miR-145 and could reverse the promotion effect of miR-145 on the progression of AAD.
CONCLUSION
miR-145 suppressed the progression of AAD by targeting CTGF, suggesting that a miR-145/CTGF axis may provide a potential therapeutic target for AAD.

Keyword

miR-145; acute aortic dissection; progression; CTGF

MeSH Terms

Animals
Apoptosis
Blotting, Western
Connective Tissue Growth Factor
Humans
Luciferases
MicroRNAs
Muscle, Smooth, Vascular
Rats
Tissue Donors
Connective Tissue Growth Factor
Luciferases
MicroRNAs

Figure

• 

  Fig. 1 miR-145 was decreased in human AAD specimens. (A) qRT-PCR results revealed decreased expression of miR-145 in AAD specimens, compared with NAD specimens (n=60). (B) Kaplan-Meier survival analysis showed a negative correlation of miR-145 with the survival time of aortic dissection patients (n=60). *p<0.05. AAD, acute aortic dissection; NAD, normal aortic dissection.

• 

  Fig. 2 miR-145 mimic promotes VSMC proliferation and inhibits VSMC apoptosis. (A) miR-145 expression was increased in VSMCs transfected with miR-145 mimic or decreased with miR-145 inhibitor. (B) VSMC viability increased after treating with miR-145 mimic or decreased after treating with miR-145 inhibitor. (C and D) Caspase-8 expression was inhibited by miR-145 mimic and promoted by miR-145 inhibitor in VSMCs according to Western blot and qRT-PCR. *p<0.05, †p<0.01. VSMC, vascular smooth muscle cell.

• 

  Fig. 3 CTGF is a target of miR-145 in VSMCs. (A) miR-145 was bound to the 3′UTR of CTGF. (B) The luciferase activity of CTGF was decreased by miR-145 mimic in WT. (C) Western blot analysis showed higher expression of CTGF protein in AAD specimens than in NAD specimens. (D) qRT-PCR showed a higher expression of CTGF mRNA in AAD specimens than in NAD specimens. (E) Regression analysis showed a negative correlation of miR-145 with CTGF expression. (F) Western blot showed decreased or increased expression of CTGF protein in the miR-145 mimic or miR-145 inhibitor group, respectively. (G) qRT-PCR showed decreased or increased expression of CTGF gene in the miR-145 mimic or miR-145 inhibitor group, respectively. *p<0.05, †p<0.01. CTGF, connective tissue growth factor; VSMC, vascular smooth muscle cell; AAD, acute aortic dissection; NAD, normal aortic dissection.

• 

  Fig. 4 CTGF attenuates miR-145 effects on VSMC proliferation and apoptosis. (A) qRT-PCR showed declined expression of CTGF in the CTGF siRNA group. (B) VSMC viability was detected by MTT for cells transfected with miR-145 inhibitor, CTGF siRNA, or both miR-145 inhibitor and CTGF siRNA. (C) Protein expression of Caspase-8 was detected by Western blot after cells were transfected with miR-145 inhibitor, CTGF siRNA, or both miR-145 inhibitor and CTGF siRNA. (D) mRNA expression of Caspase-8 was detected by qRT-PCR after cells were transfected with miR-145 inhibitor, CTGF siRNA, or both miR-145 inhibitor and CTGF siRNA. *p<0.05, †p<0.01. CTGF, connective tissue growth factor; VSMC, vascular smooth muscle cell.

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