title: PlGF gene knockdown in human retinal pigment epithelial cells.
abstract: BACKGROUND
  To evaluate the knockdown of placental growth factor (PlGF) gene expression in human retinal pigment epithelium (RPE) cells and its effect on cell proliferation, apoptosis and angiogenic potential of RPE cells.
  
  
  METHODS
  Human RPE cells were isolated by dispase I solution and cultured in DMEM/F12 supplemented with 10% fetal calf serum (FCS). A small interfering RNA (siRNA) corresponding to PlGF mRNA and a scrambled siRNA (scRNA) were introduced into the cells. Cell proliferation and cell death were examined by ELISA. PlGF mRNA and protein were quantified by real-time polymerase chain reaction (PCR) and western blot. The levels of gene expression for human retinal pigment epithelium-specific protein 65 kDa (RPE65), cellular retinaldehyde-binding protein (CRALBP) and tyrosinase were examined by real-time PCR. The angiogenic activity of RPE cell-derived conditioned media was assayed by a tube formation assay using human umbilical vein endothelial cells (HUVECs).
  
  
  RESULTS
  At a final siRNA concentration of 20 pmol/ml, the transfection efficiency was about 80%. The amount of PlGF transcripts was reduced to 10% after 36 h of incubation, and the amount of PlGF protein in culture supernatant was significantly decreased. Suppression of PlGF gene had no effect on RPE cell proliferation and survival, and there were no notable changes in the transcript levels of RPE65, CRALBP or tyrosinase for the cultures treated by siRNA cognate to PlGF. Vascular tube formation was efficiently reduced in HUVECs.
  
  
  CONCLUSIONS
  Our findings present PlGF as a key modulator of angiogenic potential in RPE cells of the human retina.
authors.affiliation: Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Hassan
authors.initials: H
authors.lastname: Akrami
authors.forename: Zahra-Soheila
authors.initials: ZS
authors.lastname: Soheili
authors.forename: Majid
authors.initials: M
authors.lastname: Sadeghizadeh
authors.forename: Hamid
authors.initials: H
authors.lastname: Ahmadieh
authors.forename: Mozhgan
authors.initials: M
authors.lastname: Rezaeikanavi
authors.forename: Shahram
authors.initials: S
authors.lastname: Samiei
authors.forename: Keynoush
authors.initials: K
authors.lastname: Khalooghi
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics; D049109:Cell Proliferation; D002478:Cells, Cultured; D004730:Endothelium, Vascular; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D055785:Gene Knockdown Techniques; D020868:Gene Silencing / Q000502:physiology*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D014442:Monophenol Monooxygenase / Q000235:genetics; D009389:Neovascularization, Pathologic / Q000517:prevention & control; D000072483:Placenta Growth Factor; D011257:Pregnancy Proteins / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
chemical_list: D002352:Carrier Proteins; D005136:Eye Proteins; D014442:Monophenol Monooxygenase; D000072483:Placenta Growth Factor; D011257:Pregnancy Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C020932:11-cis-retinal-binding protein; C000608036:PGF protein, human; C467484:VEGFA protein, human