Study On TGF-?/Smad Signaling Pathway In Lens Epithelial Cells

Objective:Posterior capsule opacification (PCO) is the most common post-operative complication of cataract surgery causing visual loss. Currently, increasing attention is directed at the TGF-B/Smad signaling pathways in lens epithelial cells. The signaling pathway of TGF-?2/Smad plays an important role in the pathological process in posterior capsule opacification after cataract surgery. Smad2 and Smad3 are both receptor-regulated Smads (R-Smads) of the TGF-?2 signaling pathway. We used RNA interference technique to inhibit TGF-B2/Smad signaling pathway. We constructed Smad2 and Smad3 siRNA expression vectors and investigate whether knockdown of Smad2, Smad3, or Smad2&3 plays a key role in PCO pathology. The signal characteristics of TGF-?2 and Smad proteins in the human lens cell line HLE-B3 were investigated.Methods:1. We designed Smad2 and Smad3 siRNA of human and cloned Smad2 and Smad3 siRNA into the eukaryotic expression vector pSilencer2.1-U6neo to construct the recombinant plasmid. After transformed into E.coli cells, the recombinant plasmids were confirmed by sequence analysis and restrict enzyme digestion.2. Smad2, Smad3, or Smad2&3 were silenced using small interfering RNA. We then tested cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell growth curve assays, migration by transwell and wound-healing assays, and extracellular matrix production including a-smooth muscle actin (aSMA), fibronectin, and type?collagen by real-time PCR assay.Results:1. The recombinant plasmids were confirmed by sequence analysis and restrict enzyme digestion. The plasmids were constructed successfully.2. With and without TGF-?2 exposure, silence of Smad3 blocked the effect of TGF-?2 on cell proliferation and production of fibronectin and type?collagen. Silence of Smad2 blocked the effect of TGF-?2 on cell migration and production of aSMA. Smad2 depletion enhanced Smad3 activity in cell proliferation and ECM production, whereas Smad3 depletion enhanced Smad2 activity in migration and aSMA expression. Silence of Smad2 and Smad3 efficiently blocked the effect of TGF-?2?on cell proliferation, migration, and extracellular matrix production. Smad2 and Smad3 are both key in the TGF-?2 signaling pathway.Conclusion:1. Smad2 and Smad3 siRNA can effectively inhibit TGF-?2/Smad signaling pathway and the expression of Smad2 and Smad3 mRNA and protein.2. Smad2 mediates the effect of TGF-?2 on cell migration and production of aSMA. Smad3 mediates TGF-?2's effect on cell proliferation and production of ECM. Smad2 depletion enhances Smad3 activity, and vice versa. These data provide a clue to prevent the development of PCO following cataract surgery by blocking the TGF-B2/Smad2&3 signaling pathway.