title: Effect of miR-20b on Apoptosis, Differentiation, the BMP Signaling Pathway and Mitochondrial Function in the P19 Cell Model of Cardiac Differentiation In Vitro.
abstract: OBJECTIVE
  To explore the effect of miR-20b on apoptosis, differentiation, the BMP signaling pathway and mitochondrial function in the P19 cell model of cardiac differentiation in vitro.
  
  
  METHODS
  A miR-20b over-expression vector, a miR-20b silencing vector and their corresponding empty vectors were constructed and transfected into P19 cells, separately. Stably miR-20b overexpressing and silenced P19 cell lines were successfully selected by blasticidin and puromycin, separately. The cells were induced to undergo apoptosis in FBS-free-α-MEM. The induced cells were examined by flow cytometry and measurement of their caspase-3 activities. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate the relative expression of marker genes of cardiomyocytes during differentiation, such as cTnT, GATA4 and ANP. QRT-PCR was also used to detect the mitochondrial DNA (mtDNA) copy number. We investigated the cellular ATP production using a luciferase-based luminescence assay. The reactive oxygen species (ROS) was determined by DCFDA (2', 7'-Dichlorofluorescein diacetate) and the mitochondrial membrane potential (MMP) was elucidated by a JC-1 fluorescent probe, both using fluorescence microscopy and flow cytometer. The expression of BMP signaling pathway-related proteins were analyzed by Western blotting.
  
  
  RESULTS
  Stably miR-20b overexpressing and silenced P19 cell lines were successfully obtained. MiR-20b overexpression increased apoptosis and promoted differentiation in P19 cells by promoting the activation of the BMP signaling pathway. In addition, miR-20b overexpression induced mitochondrial impairment in P19 cells during differentiation, which was characterized by lower MMP, raised ATP synthesis and increased ROS levels. The effects of miR-20b silencing were the exact opposite to those of overexpression.
  
  
  CONCLUSIONS
  Collectively, these results suggested that miR-20b was very important in apoptosis, differentiation and mitochondrial function of P19 cells. MiR-20b may represent a new therapeutic target for congenital heart diseases and provide new insights into the mechanisms of cardiac diseases.
authors.affiliation: Department of Cardiology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China.
authors.forename: Shasha
authors.initials: S
authors.lastname: Zhu
authors.affiliation: State key Laboratory of Reproductive Medicine, Department of Pediatrics, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210004, China.
authors.forename: Xiaoshan
authors.initials: X
authors.lastname: Hu
authors.affiliation: State key Laboratory of Reproductive Medicine, Department of Pediatrics, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210004, China.
authors.forename: Zhangbin
authors.initials: Z
authors.lastname: Yu
authors.affiliation: State key Laboratory of Reproductive Medicine, Department of Pediatrics, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210004, China.
authors.forename: Yuzhu
authors.initials: Y
authors.lastname: Peng
authors.affiliation: State key Laboratory of Reproductive Medicine, Department of Pediatrics, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210004, China.
authors.forename: Jingai
authors.initials: J
authors.lastname: Zhu
authors.affiliation: State key Laboratory of Reproductive Medicine, Department of Pediatrics, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210004, China.
authors.forename: Xuehua
authors.initials: X
authors.lastname: Liu
authors.affiliation: State key Laboratory of Reproductive Medicine, Department of Pediatrics, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210004, China.
authors.forename: Mengmeng
authors.initials: M
authors.lastname: Li
authors.affiliation: State key Laboratory of Reproductive Medicine, Department of Pediatrics, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210004, China.
authors.forename: Shuping
authors.initials: S
authors.lastname: Han
authors.affiliation: State key Laboratory of Reproductive Medicine, Department of Pediatrics, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210004, China.
authors.forename: Chun
authors.initials: C
authors.lastname: Zhu
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D017209:Apoptosis; D019485:Bone Morphogenetic Proteins / Q000502:physiology*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D048430:Cell Shape; D015870:Gene Expression; D006330:Heart Defects, Congenital / Q000235:genetics / Q000628:therapy; D053078:Membrane Potential, Mitochondrial; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000502:physiology*; D008928:Mitochondria / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000502:physiology; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction
chemical_list: C485672:Bambi protein, mouse; D019485:Bone Morphogenetic Proteins; D008565:Membrane Proteins; D035683:MicroRNAs; C532375:Mirn20 microRNA, mouse; D017382:Reactive Oxygen Species; D000255:Adenosine Triphosphate