Small Interfering RNA Identifies the Role of p57KIP2 and p15INK4b in TGF–ß1 Inhibited Proliferation of Primary Cultured Human Corneal Epithelial Cells

Abstract

Abstract: : Purpose: Small interfering RNA (siRNA) has become a powerful tool for silencing gene expression and function in mammalian cells. This work was to evaluate a role of cyclin–dependent kinase (CDK) inhibitors, p57KIP2 (p57) and p15INK4b (p15), in TGFß1 inhibited proliferation of primary cultured human corneal epithelial cells using siRNA. Methods: Primary cultured human corneal epithelial cells were treated with 1ng/ml of TGFß1 for 6 and 24 hours, and subjected to total RNA extraction for RT–PCR with primers for CDK inhibitors, p21, p27 and p57 (CIP/KIP family) and p15 and p19 (INK4 family). The effect of TGF–ß1 on cell proliferation was evaluated by BrdU incorporation and colony forming efficiency (CFE) on a mouse 3T3 fibroblast feeder layer. For RNA interference assay, primary cultures were passaged at 4x10⁴ cells/cm² into 12–well plates in Keratinocytes Serum–Free Medium for 24 hours, and transfected by annealed double–stranded siRNA (2 µg/well) for p57, p15, or siRNA–Fluorescein (siRNA–F) as a negative control with Qiagen RNAiFect reagent for 48 hours, followed by treatment with or without adding TGFß1 for additional 6–24 hours. Results: TGFß1 significantly inhibited the cell proliferation showing a decreased BrdU incorporation and CFE. TGFß1 up–regulated the expression of p57 and p15 mRNA, while did not effect the expression of p19, p21 and p27 after treatment for 6 and 24 hours. About 80% cells were transfected with these siRNAs in 6 hours without visible cell damage, as indicated by siRNA–F and cell morphology. The TGF–ß1 stimulated expression of p57 and p15 mRNA was markedly blocked by siRNA–p57 or –p15, respectively, but not by siRNA–F. The TGFß1 suppressed BrdU incorporation was increased to near normal by siRNA–p57 or –p15. Conclusions: These findings demonstrate that the siRNA can be successfully transfected into primary cultured human corneal epithelial cells and temporally knocked down the target gene. The siRNA targeted to p57KIP2 and p15INK4b identifies their role in the inhibitory effect of TGF–ß1 on epithelial cell growth.

Keywords: cornea: epithelium • gene transfer/gene therapy • signal transduction