Altered microRNAs in bicuspid aortic valve: a comparison between stenotic and insufficient valves.
OBJECTIVE
Bicuspid aortic valve (BAV), the most common form of congenital heart disease, is a leading cause of aortic stenosis (AS) and aortic insufficiency (AI). AS is typically caused by calcific valve disease. Recently, microRNAs (miRNAs) have been shown to modulate gene expression. The study aim was to examine the miRNAs that were altered in the aortic valve leaflets of patients with AS compared to those in patients with AI. In-vitro experiments were also carried out to determine if these miRNAs could modulate calcification-related genes.


METHODS
Aortic valve samples (fused and unfused leaflets) were collected from nine male patients (mean age 44.9 +/- 13.8 years) undergoing aortic valve replacement (AVR). PIQOR miRXplore Microarrays containing 1,421 miRNAs were used and hybridized to fused leaflet samples labeled with Cy5; unfused samples were used as controls and labeled with Cy3. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to validate the miRNA array results. Cultured human aortic valve interstitial cells (AVICs) were treated with miRNA mimics, and qRT-PCR was carried out to determine any changes in mRNAs.


RESULTS
By microarray analysis, seven miRNAs were shown to be statistically different between the AS and AI patients. In the stenotic samples, the MiR-26a and miR-195 levels were shown (by qRT-PCR) to be reduced by 65% and 59%, respectively (p < 0.05), and MiR-30b to be reduced by 62% (p < 0.06). Human AVICs treated with miR-26a or miR-30b mimics showed decreased mRNA levels of calcification-related genes. MiR-26a repressed BMP2 by 36%, alkaline phosphatase (ALPL) by 38%, and SMAD1 by 26%, while MiR-30b reduced the expression of SMAD1 by 18% and of SMAD3 by 12%. In contrast, miR-195-treated AVICs had increased mRNA levels of calcification-related genes, such as BMP2 by 68% and RUNX2 by 11%.


CONCLUSIONS
MiR-26a, miR-30b, and miR-195 were each decreased in the aortic valves of patients requiring AVR due to AS, compared to those requiring replacement due to AI. These miRNAs appear to modulate calcification-related genes in vitro.
Nigam
Vishal
V
Department of Pediatrics (Cardiology), University of California, San Francisco, USA.
Sievers
Hans H
HH
Jensen
Brian C
BC
Sier
Holger A
HA
Simpson
Paul C
PC
Srivastava
Deepak
D
Mohamed
Salah A
SA
D000328:Adult; D001021:Aortic Valve / Q000002:abnormalities / Q000378:metabolism* / Q000601:surgery; D001022:Aortic Valve Insufficiency / Q000235:genetics* / Q000601:surgery; D001024:Aortic Valve Stenosis / Q000235:genetics* / Q000601:surgery; D002114:Calcinosis / Q000235:genetics* / Q000601:surgery; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D005819:Genetic Markers; D006330:Heart Defects, Congenital / Q000235:genetics* / Q000601:surgery; D019918:Heart Valve Prosthesis Implantation; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000378:metabolism*; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012495:San Francisco
D005819:Genetic Markers; D035683:MicroRNAs; D012333:RNA, Messenger; C528163:MIRN26 microRNA, human