title: Triple combination of siRNAs targeting TGFβ1, TGFβR2, and CTGF enhances reduction of collagen I and smooth muscle actin in corneal fibroblasts.
abstract: OBJECTIVE
  Transforming growth factor β1 (TGFβ1), TGFβ receptor (TGFβR2), and connective tissue growth factor (CTGF) are key regulators of fibrosis in the cornea and in other tissues, including liver, skin, and kidney. We developed an antifibrotic treatment targeting these three critical scarring genes by using a combination of small interfering RNAs (siRNAs) and assessed its effect on downstream scarring genes, collagen I, and α smooth muscle actin (SMA).
  
  
  METHODS
  Up to six individual siRNAs for each of the three target gene mRNAs were transfected into cultures of rabbit corneal fibroblasts at concentrations from 15 to 90 nM. The knockdown of target gene proteins was measured by ELISA, and the two most effective siRNAs were tested in dual combinations. Knockdown percentages of both individual and dual siRNA combinations were analyzed for synergy by using combination index to predict "effective" and "ineffective" triple siRNA combinations. Effects of both triple siRNA combinations on target and downstream mRNAs were measured by using quantitative RT-PCR, and levels of SMA protein were assessed by immunohistochemistry.
  
  
  RESULTS
  Single and dual siRNA combinations produced a wide range of protein knockdown of target genes (5%-80%). The effective triple siRNA combination significantly reduced mRNA levels of target genes (>80%) and downstream scarring genes (>85%), and of SMA protein (>95%), and significantly reduced cell migration without reducing cell viability.
  
  
  CONCLUSIONS
  Simultaneous targeting of TGFβ1, TGFβR2, and CTGF genes by effective triple siRNA combination produced high knockdown of target and downstream scarring genes without cell toxicity, which may have clinical applications in reducing corneal fibrosis and scarring in other tissues.
authors.affiliation: Department of Obstetrics and Gynecology, Institute for Wound Research, University of Florida, Gainesville, Florida.
authors.forename: Sriniwas
authors.initials: S
authors.lastname: Sriram
authors.forename: Paulette
authors.initials: P
authors.lastname: Robinson
authors.forename: Liya
authors.initials: L
authors.lastname: Pi
authors.forename: Alfred S
authors.initials: AS
authors.lastname: Lewin
authors.forename: Gregory
authors.initials: G
authors.lastname: Schultz
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D055513:Connective Tissue Growth Factor / Q000235:genetics* / Q000378:metabolism; D003315:Cornea / Q000378:metabolism* / Q000473:pathology; D003316:Corneal Diseases / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D015316:Genetic Therapy / Q000379:methods; D007150:Immunohistochemistry; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053773:Transforming Growth Factor beta1 / Q000235:genetics* / Q000378:metabolism; D053781:Transforming Growth Factor beta2 / Q000235:genetics* / Q000378:metabolism
chemical_list: D000199:Actins; D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D055513:Connective Tissue Growth Factor; D003094:Collagen
keywords: CTGF; RNA interference; TGFβ1; combination therapy; siRNA