title: Knockdown of Plakophilin 2 Downregulates miR-184 Through CpG Hypermethylation and Suppression of the E2F1 Pathway and Leads to Enhanced Adipogenesis In Vitro.
abstract: BACKGROUND
  PKP2, encoding plakophilin 2 (PKP2), is the most common causal gene for arrhythmogenic cardiomyopathy.
  
  
  OBJECTIVE
  To characterize miRNA expression profile in PKP2-deficient cells.
  
  
  RESULTS
  Control and PKP2-knockdown HL-1 (HL-1(Pkp2-shRNA)) cells were screened for 750 miRNAs using low-density microfluidic panels. Fifty-nine miRNAs were differentially expressed. MiR-184 was the most downregulated miRNA. Expression of miR-184 in the heart and cardiac myocyte was developmentally downregulated and was low in mature myocytes. MicroRNA-184 was predominantly expressed in cardiac mesenchymal progenitor cells. Knockdown of Pkp2 in cardiac mesenchymal progenitor cells also reduced miR-184 levels. Expression of miR-184 was transcriptionally regulated by the E2F1 pathway, which was suppressed in PKP2-deficient cells. Activation of E2F1, on overexpression of its activator CCND1 (cyclin D1) or knockdown of its inhibitor retinoblastoma 1, partially rescued miR-184 levels. In addition, DNA methyltransferase-1 was recruited to the promoter region of miR-184, and the CpG sites at the upstream region of miR-184 were hypermethylated. Treatment with 5-aza-2'-deoxycytidine, a demethylation agent, and knockdown of DNA methyltransferase-1 partially rescued miR-184 level. Pathway analysis of paired miR-184:mRNA targets identified cell proliferation, differentiation, and death as the main affected biological processes. Knockdown of miR-184 in HL-1 cells and mesenchymal progenitor cells induced and, conversely, its overexpression attenuated adipogenesis.
  
  
  CONCLUSIONS
  PKP2 deficiency leads to suppression of the E2F1 pathway and hypermethylation of the CpG sites at miR-184 promoter, resulting in downregulation of miR-184 levels. Suppression of miR-184 enhances and its activation attenuates adipogenesis in vitro. Thus, miR-184 contributes to the pathogenesis of adipogenesis in PKP2-deficient cells.
authors.affiliation: From the Center for Cardiovascular Genetics, Institute of Molecular Medicine and Department of Medicine, University of Texas Health Sciences Center at Houston, and Texas Heart Institute.
authors.forename: Priyatansh
authors.initials: P
authors.lastname: Gurha
authors.affiliation: From the Center for Cardiovascular Genetics, Institute of Molecular Medicine and Department of Medicine, University of Texas Health Sciences Center at Houston, and Texas Heart Institute.
authors.forename: Xiaofan
authors.initials: X
authors.lastname: Chen
authors.affiliation: From the Center for Cardiovascular Genetics, Institute of Molecular Medicine and Department of Medicine, University of Texas Health Sciences Center at Houston, and Texas Heart Institute.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Lombardi
authors.affiliation: From the Center for Cardiovascular Genetics, Institute of Molecular Medicine and Department of Medicine, University of Texas Health Sciences Center at Houston, and Texas Heart Institute.
authors.forename: James T
authors.initials: JT
authors.lastname: Willerson
authors.affiliation: From the Center for Cardiovascular Genetics, Institute of Molecular Medicine and Department of Medicine, University of Texas Health Sciences Center at Houston, and Texas Heart Institute. Ali.J.Marian@uth.tmc.edu Priyatansh.Gurha@uth.tmc.edu.
authors.forename: Ali J
authors.initials: AJ
authors.lastname: Marian
mesh_terms: D050156:Adipogenesis / Q000502:physiology*; D000818:Animals; D002478:Cells, Cultured; D018899:CpG Islands / Q000502:physiology*; D019175:DNA Methylation / Q000502:physiology*; D015536:Down-Regulation / Q000502:physiology; D050687:E2F1 Transcription Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D035683:MicroRNAs / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism; D051191:Plakophilins / Q000172:deficiency* / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology
chemical_list: D050687:E2F1 Transcription Factor; C493754:E2f1 protein, mouse; C552233:MIRN184 microRNA, mouse; D035683:MicroRNAs; D051191:Plakophilins
keywords: DNA methylation; cardiomyopathy; epigenetics; gene expression; heart failure; microRNA