title: Altered DNA Methylation of Long Noncoding RNA H19 in Calcific Aortic Valve Disease Promotes Mineralization by Silencing NOTCH1.
abstract: BACKGROUND
  Calcific aortic valve disease is characterized by an abnormal mineralization of the aortic valve. Osteogenic activity in the aortic valve is under the control of NOTCH1, which regulates the expression of key pro-osteogenic genes such as RUNX2 and BMP2. Long noncoding RNAs (lncRNAs) may reprogram cells by altering the gene expression pattern.
  
  
  METHODS
  Multidimensional genomic profiling was performed in human aortic valves to document the expression of lncRNAs and the DNA methylation pattern in calcific aortic valve disease. In-depth functional assays were carried out to document the impact of lncRNA on the mineralization of the aortic valve.
  
  
  RESULTS
  We documented that lncRNA H19 (H19) was increased in calcific aortic valve disease. Hypomethylation of the promoter region was observed in mineralized aortic valves and was inversely associated with H19 expression. Knockdown and overexpression experiments showed that H19 induces a strong osteogenic phenotype by altering the NOTCH1 pathway. Gene promoter analyses showed that H19 silenced NOTCH1 by preventing the recruitment of p53 to its promoter. A knockdown of H19 in valve interstitial cells (VICs) increased the expression of NOTCH1 and decreased the level of RUNX2 and BMP2, 2 downstream targets repressed by NOTCH1. In rescue experiments, the transfection of a vector encoding for the active Notch intracellular domain prevented H19-induced mineralization of valve interstitial cells.
  
  
  CONCLUSIONS
  These findings indicate that a dysregulation of DNA methylation in the promoter of H19 during calcific aortic valve disease is associated with a higher expression of this lncRNA, which promotes an osteogenic program by interfering with the expression of NOTCH1.
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.).
authors: Fayez
authors: F
authors: Hadji
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.).
authors: Marie-Chloé
authors: MC
authors: Boulanger
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.).
authors: Simon-Pierre
authors: SP
authors: Guay
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.).
authors: Nathalie
authors: N
authors: Gaudreault
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.).
authors: Soumiya
authors: S
authors: Amellah
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.).
authors: Guada
authors: G
authors: Mkannez
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.).
authors: Rihab
authors: R
authors: Bouchareb
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.).
authors: Joël Tremblay
authors: JT
authors: Marchand
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.).
authors: Mohamed Jalloul
authors: MJ
authors: Nsaibia
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.).
authors: Sandra
authors: S
authors: Guauque-Olarte
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.).
authors: Philippe
authors: P
authors: Pibarot
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.).
authors: Luigi
authors: L
authors: Bouchard
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.).
authors: Yohan
authors: Y
authors: Bossé
authors: From Laboratory of Cardiovascular Pathobiology, Quebec Heart and Lung Institute/Research Center, Department of Surgery (F.H., M.-C.B, N.G., S.A., G.M., R.B., M.J.N., P.M.), Department of Molecular Medicine (J.T.M., S.G.-O., Y.B.), and Department of Medicine (P.P.), Laval University, Quebec, QC, Canada; Department of Biochemistry, Université de Sherbrooke, Sherbrooke, QC, Canada (S.-P.G., L.B.); and ECOGENE-21 and Lipid Clinic, Chicoutimi Hospital, Saguenay, QC, Canada (S.-P.G., L.B.). patrick.mathieu@fmed.ulaval.ca yohan.bosse@criucpq.ulaval.ca.
authors: Patrick
authors: P
authors: Mathieu
mesh_terms: D000368:Aged; D001021:Aortic Valve / Q000166:cytology / Q000378:metabolism / Q000473:pathology*; D001024:Aortic Valve Stenosis / Q000235:genetics* / Q000473:pathology; D055396:Bone Morphogenetic Protein 2 / Q000032:analysis; D002114:Calcinosis / Q000235:genetics* / Q000473:pathology; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D019175:DNA Methylation; D005260:Female; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D062085:RNA, Long Noncoding / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D051881:Receptor, Notch1 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000032:analysis
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D050659:Core Binding Factor Alpha 1 Subunit; C094335:H19 long non-coding RNA; C497761:NOTCH1 protein, human; D062085:RNA, Long Noncoding; D034741:RNA, Small Interfering; D051881:Receptor, Notch1; D016159:Tumor Suppressor Protein p53
keywords: H19 long non-coding RNA; RNA, long noncoding; aortic valve; aortic valve stenosis; aortic valve, calcification of; core binding factor alpha 1 subunit; receptor, notch1