title: Depletion of SLC4A11 causes cell death by apoptosis in an immortalized human corneal endothelial cell line.
abstract: OBJECTIVE
  To investigate the effects of SLC4A11 gene depletion in human corneal endothelial cells.
  
  
  METHODS
  To achieve stable downregulation of SLC4A11 gene expression in immortalized human corneal endothelial cells (HCECs), short-hairpin RNA (shRNA) targeted against SLC4A11 was used. Cell growth and viability were determined using the real-time cell analyzer and trypan blue staining respectively. Apoptosis was investigated by Annexin V and TUNEL assays. Alterations in apoptotic gene expression following SLC4A11 silencing were determined using the RT(2)Profiler PCR array for human apoptosis while activation of the apoptotic pathway was ascertained by western analysis.
  
  
  RESULTS
  SLC4A11 silencing in HCECs could be achieved by stable expression of shRNA targeted against SLC4A11. SLC4A11 knockdown suppressed HCEC growth and reduced HCEC viability compared to the control. This reduction in cell growth is associated with increased apoptosis in SLC4A11-silenced cells.
  
  
  CONCLUSIONS
  Our data suggest that the reduction of cell number with time in SLC4A11-depleted HCECs is due to an increase in cell death by apoptosis. This suggests that SLC4A11 is necessary for cell survival and may explain the pathologic corneal endothelial cell loss in endotheliopathies due to SLC4A11 mutations.
authors.affiliation: Singapore Eye Research Institute, Singapore.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Li-Fong
authors.initials: LF
authors.lastname: Seet
authors.forename: Li Wei
authors.initials: LW
authors.lastname: Koh
authors.forename: Anandalakshmi
authors.initials: A
authors.lastname: Venkatraman
authors.forename: Divya
authors.initials: D
authors.lastname: Venkataraman
authors.forename: Rajiv R
authors.initials: RR
authors.lastname: Mohan
authors.forename: Jeppe
authors.initials: J
authors.lastname: Praetorius
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Bonanno
authors.forename: Tin
authors.initials: T
authors.lastname: Aung
authors.forename: Eranga N
authors.initials: EN
authors.lastname: Vithana
mesh_terms: D027321:Anion Transport Proteins / Q000096:biosynthesis / Q000235:genetics*; D017920:Antiporters / Q000096:biosynthesis / Q000235:genetics*; D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D002452:Cell Count; D016923:Cell Death / Q000235:genetics*; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D015536:Down-Regulation / Q000235:genetics*; D004728:Endothelium, Corneal / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics*; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D027321:Anion Transport Proteins; D017920:Antiporters; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C425809:SLC4A11 protein, human