title: [Effects of Gremlin on transdifferentiation and extracellular matrix synthesis in human lens epithelium cells].
abstract: OBJECTIVE
  To explore the effects of Gremlin on transdifferentiation and extracellular matrix synthesis in cultured human lens epithelium cells (HLEC).
  
  
  METHODS
  This is an experimental research. HLEC were incubated with different concentrations of Gremlin (0, 100, 200 and 400 µg/L) for 24 h. The morphological changes of HLEC were observed by inverted microscope. Real-time PCR and Western-Blot were used to evaluate the expression of α-smooth muscle actin (α-SMA) (as a landmark protein of epithelial mesenchymal transition), fibronectin (Fn) and collagen type 1 (COL-1) (as major components of extracellular matrix) after stimulation with different time (0, 12, 24, 48, 72 h) by 200 µg/L Gremlin. The same parameters were observed in Gremlin. siRNA transfected HLEC which treated with 1.0 µg/L TGF-β2. α-SMA, Fn and COL-1 protein and mRNA expressions comparison with control group were analyzed using one-way and two-way ANOVA, while the difference between groups were compared using Turkey HSD and LSD-t test.
  
  
  RESULTS
  The normal morphology of HLEC showed polygonal and anchorage-dependent. After the incubation of different concentrations of Gremlin for 24 h, morphological feature of HLEC were changed from monolayer and polygonal to multilayer and spindle fibroblast-like cells, and the intercellular space widened. The expression of α-SMA, Fn and COL-1 were increased with prolonging of Gremlin treatment time (α-SMA gene induction: 1.00 ± 0.00, 1.62 ± 0.57, 3.40 ± 0.83, 5.90 ± 0.49, 7.97 ± 0.91; F = 61.64, P < 0.05, q = 6.43, 13.13, 18.66, P < 0.05; Fn gene induction: 1.00 ± 0.00, 3.26 ± 0.23, 5.86 ± 0.90, 10.17 ± 2.16, 12.89 ± 1.63; F = 42.03, P < 0.05, q = 6.45, 12.18, 15.79, P < 0.05; COL-1 gene induction: 1.00 ± 0.00, 1.78 ± 0.88, 6.80 ± 0.44, 12.76 ± 2.46, 21.12 ± 3.66; F = 51.79, P < 0.05, q = 4.97, 10.08, 17.26, P < 0.05) (α-SMA protein expression: 0.13 ± 0.02, 0.26 ± 0.02, 0.29 ± 0.09, 0.47 ± 0.06, 0.68 ± 0.05; F = 45.14, q = 5.11, 10.67, 17.40, P < 0.05; Fn protein expression: 0.16 ± 0.04, 0.26 ± 0.07, 0.65 ± 0.03, 0.82 ± 0.04, 0.73 ± 0.02; F = 144.4, q = 20.09, 26.78, 23.12, P < 0.05; COL-1 protein expression: 0.11 ± 0.02, 0.23 ± 0.09, 0.41 ± 0.05, 0.61 ± 0.03, 0.74 ± 0.03; F = 75.47, q = 9.99, 16.60, 21.07, P < 0.05). Gremlin. siRNA transfection effectively suppressed TGF-β2-induced expression of α-SMA, Fn, and COL-I (α-SMA gene induction: F = 81.89, P < 0.05, t = 3.234, 4.346, 10.35; t = 2.252, 7.272, 19.11, P < 0.05; Fn gene induction: F = 83.61, P < 0.05, t = 2.538, 8.202, 11.99; t = 6.316, 7.304, 14.80, P < 0.05; COL-1 gene induction: F = 73.64, P < 0.05, t = 3.385, 7.942, 11.64; t = 4.794, 9.006, 13.75, P < 0.05; Gremlin gene induction: F = 46.11, P < 0.05, t = 5.08, 7.24, 8.27; t = 6.27, 8.27, 12.14, P < 0.05) (α-SMA protein expression: F = 129.6, P < 0.05, t = 4.34, 4.85; 3.83, 4.34; 13.03, 14.82, P < 0.05; Fn protein expression: F = 26.18, P < 0.05, t = 5.68, 5.95; 3.10, 4.06; 4.19, 4.73, P < 0.05; COL-1 protein expression: F = 41.37, P < 0.05, t = 6.93, 5.51; 7.82, 6.93; 8.71, 7.64, P < 0.05; Gremlin protein expression: F = 59.52, P < 0.05, t = 2.24, 3.49; 5.74, 6.23; 6.98, 9.98, P < 0.05).
  
  
  CONCLUSIONS
  Gremlin could induce HLEC to express α-SMA, Fn and COL-1 in a time-dependent manner and promote transdifferentiation and extracellular matrix synthesis. Specifically silencing the expression of Gremlin could effectively block the TGF-β2-induced EMT and ECM synthesis in HLEC.
authors.affiliation: Department of Ophthalmology, the First Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an 710061, China; Email: peich71@163.com.
authors.forename: Cheng
authors.initials: C
authors.lastname: Pei
authors.affiliation: Department of Ophthalmology, the First Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Ma
authors.affiliation: Department of Ophthalmology, the First Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Qianyan
authors.initials: Q
authors.lastname: Kang
authors.affiliation: Department of Ophthalmology, the First Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Li
authors.initials: L
authors.lastname: Qin
authors.affiliation: Department of Ophthalmology, the First Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Lijun
authors.initials: L
authors.lastname: Cui
mesh_terms: D000199:Actins / Q000378:metabolism; D002460:Cell Line; D054338:Cell Transdifferentiation / Q000187:drug effects*; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects* / Q000502:physiology; D058750:Epithelial-Mesenchymal Transition; D005109:Extracellular Matrix / Q000187:drug effects* / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000166:cytology*; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D053781:Transforming Growth Factor beta2
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D024042:Collagen Type I; D005353:Fibronectins; C486399:GREM1 protein, human; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D053781:Transforming Growth Factor beta2