title: Systems biology approach to transplant tolerance: proof of concept experiments using RNA interference (RNAi) to knock down hub genes in Jurkat and HeLa cells in vitro.
abstract: BACKGROUND
  Systems biology is gaining importance in studying complex systems such as the functional interconnections of human genes [1]. To investigate the molecular interactions involved in T cell immune responses, we used databases of physical gene-gene interactions to constructed molecular interaction networks (interconnections) with R language algorithms. This helped to identify highly interconnected "hub" genes AT(1)P5C1, IL6ST, PRKCZ, MYC, FOS, JUN, and MAPK1. We hypothesized that suppression of these hub genes in the gene network would result in significant phenotypic effects on T cells and examined this in vitro. The molecular interaction networks were then analyzed and visualized with Cytoscape.
  
  
  METHODS
  Jurkat and HeLa cells were transfected with siRNA for the selected hub genes. Cell proliferation was measured using ATP luminescence and BrdU labeling, which were measured 36, 72, and 96 h after activation.
  
  
  RESULTS
  Following T cell stimulation, we found a significant decrease in ATP production (P < 0.05) when the hub genes ATP5C1 and PRKCZ were knocked down using siRNA transfection, whereas no difference in ATP production was observed in siRNA transfected HeLa cells. However, HeLa cells showed a significant (P < 0.05) decrease in cell proliferation when the genes MAPK1, IL6ST, ATP5C1, JUN, and FOS were knocked down.
  
  
  CONCLUSIONS
  In both Jurkat and HeLa cells, targeted gene knockdown using siRNA showed decreased cell proliferation and ATP production in both Jurkat and HeLa cells. However, Jurkat T cells and HELA cells use different hub genes to regulate activation responses. This experiment provides proof of principle of applying siRNA knockdown of T cell hub genes to evaluate their proliferative capacity and ATP production. This novel concept outlines a systems biology approach to identify hub genes for targeted therapeutics.
authors.affiliation: Department of Medicine and Nephrology, University of California, San Diego, La Jolla, California, USA.
authors.forename: Wint Wah
authors.initials: WW
authors.lastname: Lwin
authors.forename: Ken
authors.initials: K
authors.lastname: Park
authors.forename: Matthew
authors.initials: M
authors.lastname: Wauson
authors.forename: Qin
authors.initials: Q
authors.lastname: Gao
authors.forename: Patricia W
authors.initials: PW
authors.lastname: Finn
authors.forename: David
authors.initials: D
authors.lastname: Perkins
authors.forename: Ajai
authors.initials: A
authors.lastname: Khanna
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006084:Graft Rejection / Q000235:genetics / Q000276:immunology*; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007108:Immune Tolerance / Q000235:genetics / Q000502:physiology*; D066298:In Vitro Techniques; D019169:Jurkat Cells / Q000187:drug effects / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D049490:Systems Biology; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
chemical_list: D034741:RNA, Small Interfering; D000255:Adenosine Triphosphate