title: Combined silencing of TGF-β2 and Snail genes inhibit epithelial-mesenchymal transition of retinal pigment epithelial cells under hypoxia.
abstract: BACKGROUND
  The formation of scar-like fibrous tissue in age-related macular degeneration (AMD) is associated with hypoxia. Under hypoxia, retinal pigment epithelial (RPE) cells can secret more transforming growth factor-β2 (TGF-β2), which is determined to induce epithelial-mesenchymal transition (EMT) at certain concentrations. Whether hypoxia can induce EMT by stimulating RPE cell line secrets TGF-β2 or not remains unknown. To gain a better understanding of the signaling mechanisms of fibrosis in AMD under hypoxic conditions, we investigated EMT in retinal pigment epithelial (RPE) cells and the effect of TGF-β2 and Snail in this process.
  
  
  METHODS
  Human RPE cell line (ARPE-19) was incubated with 5 % O2 for different periods of time. The expression of N-cadherin, α-smooth muscle actin (α-SMA), TGF-β2 , and Snail were determined by Western blot and real-time PCR. Cell proliferation was assessed by CCK8 kit. RNA interference was used for multi-gene silencing of TGF-β2 and Snail genes.
  
  
  RESULTS
  N-cadherin was decreased and mesenchymal cell marker α-SMA was increased after the ARPE-19 cell line was incubated with 5 % O2. Meanwhile, the proliferation capability of the cell line was increased. TGF-β2 and Snail expression were increased in a time-dependent manner under hypoxia. After multi-silencing TGF-β2 and Snail genes, N-cadherin was increased and α-SMA was reduced. Meanwhile, the proliferation of the cell line was suppressed.
  
  
  CONCLUSIONS
  Under hypoxic conditions, RPE cells undergo EMT. Endogenic TGF-β2 and Snail are involved in this process. Furthermore, knockdown of both TGF-β2 and Snail inhibited EMT to a greater extent than knockdown of either gene individually.
authors: Department of Ophthalmology, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Nangang district, Harbin, 150001, Heilongjiang, Province, People's Reepublic of China.
authors: Zhuolei
authors: Z
authors: Feng
authors: Ruishu
authors: R
authors: Li
authors: Huanqi
authors: H
authors: Shi
authors: Wenjiao
authors: W
authors: Bi
authors: Wenwen
authors: W
authors: Hou
authors: Xiaomei
authors: X
authors: Zhang
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology*; D020868:Gene Silencing / Q000502:physiology*; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D053781:Transforming Growth Factor beta2 / Q000235:genetics* / Q000378:metabolism
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D015703:Antigens, CD; C050361:CDH2 protein, human; D015820:Cadherins; D012333:RNA, Messenger; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D053781:Transforming Growth Factor beta2