title: Abdominal Aortic Aneurysm-Associated MicroRNA-516a-5p Regulates Expressions of Methylenetetrahydrofolate Reductase, Matrix Metalloproteinase-2, and Tissue Inhibitor of Matrix Metalloproteinase-1 in Human Abdominal Aortic Vascular Smooth Muscle Cells.
abstract: BACKGROUND
  MicroRNAs (miRNAs or miRs) have been highlighted to be involved in abdominal aortic aneurysm (AAA) with the emergence of recent miRNA microarray profiling studies. miR-516a-5p has been shown to be significantly overexpressed in vascular smooth muscle cells (VSMCs) from human AAA tissues from our previous microarray study, suggesting its crucial association with AAA. In addition, further bioinformatics analysis predicted methylenetetrahydrofolate reductase (MTHFR), which regulates homocysteine (Hcy) metabolism and is proposed to be a risk gene for AAA formation and to be the downregulation target of miR-516a-5p. However, the pathogenic role of miR-516a-5p in VSMCs for AAA formation remains unresolved. This study aims to investigate the role of miR-516a-5p in human VSMCs for AAA pathogenesis.
  
  
  METHODS
  miR-516a-5p was stably overexpressed and knocked down in VSMCs explant cultured from human abdominal aortic tissues by means of lentiviral system. The MTHFR protein expression was first examined by Western blotting. In addition, the protein expressions of several key components involved in AAA pathogenic features are as follows: matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-2 for elastin degradation; collagen type 1 alpha 1 for compensatory collagen synthesis; monocyte chemoattractant protein-1 for inflammation, were also evaluated. Apoptotic level of VSMCs was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling assay.
  
  
  RESULTS
  Results showed that protein expression of MTHFR was significantly downregulated on miR-516a-5p overexpression (P < 0.05) in VSMCs, whereas it was significantly upregulated on miR-516a-5p knockdown (P < 0.05). Of all the AAA key components investigated, only MMP-2 and TIMP-1 protein expressions were found altered. A significant increase in MMP-2 (P < 0.05) and decrease in TIMP-1 (P < 0.05) expressions were observed on miR-516a-5p overexpression in VSMCs. Apoptosis was not promoted on miR-516a-5p overexpression or knockdown in VSMCs.
  
  
  CONCLUSIONS
  Our findings suggested that miR-516a-5p may regulate MTHFR, MMP-2, and TIMP-1 expressions in human VSMCs, possibly promoting the disruption of Hcy metabolism and proteolytic degradation of elastin for AAA formation.
authors.affiliation: Division of Vascular Surgery, Department of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, Pokfulam, Hong Kong, China. Electronic address: cytc@hku.hk.
authors.forename: Crystal Yin Tung
authors.initials: CYT
authors.lastname: Chan
authors.affiliation: Division of Vascular Surgery, Department of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, Pokfulam, Hong Kong, China.
authors.forename: Bernice Lai Yee
authors.initials: BLY
authors.lastname: Cheuk
authors.affiliation: Division of Vascular Surgery, Department of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, Pokfulam, Hong Kong, China.
authors.forename: Stephen Wing Keung
authors.initials: SWK
authors.lastname: Cheng
mesh_terms: D000328:Adult; D001012:Aorta, Abdominal / Q000201:enzymology / Q000473:pathology; D017544:Aortic Aneurysm, Abdominal / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D017209:Apoptosis; D002478:Cells, Cultured; D004549:Elastin / Q000378:metabolism; D006710:Homocysteine / Q000378:metabolism; D006801:Humans; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D009131:Muscle, Smooth, Vascular / Q000201:enzymology* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000201:enzymology* / Q000473:pathology; D059748:Proteolysis; D034622:RNA Interference; D015398:Signal Transduction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism*; D014162:Transfection
chemical_list: C557718:MIRN516 microRNA, human; D035683:MicroRNAs; C560822:TIMP1 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D006710:Homocysteine; D004549:Elastin; C573423:MTHFR protein, human; D042965:Methylenetetrahydrofolate Reductase (NADPH2); C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2