Decreasing expression of the G1-phase inhibitors, p21Cip1 and p16INK4a, promotes division of corneal endothelial cells from older donors

PURPOSE The current studies were conducted to determine whether the cyclin-dependent kinase inhibitors, p21Cip1 (p21 cyclin-dependent kinase-interacting protein 1) and p16INK4a (p16 cyclin-dependent kinase inhibitor 1A), help mediate G(1)-phase inhibition in human corneal endothelial cells (HCEC) by testing the effect of siRNA (small interfering RNA)-mediated down-regulation of the expression of these inhibitors on cell cycle entry and proliferation in HCEC cultured from older donors. METHODS HCEC were obtained from National Disease Research Interchange, Philadelphia, PA, and cultured according to published methods. Cells were electroporated in the presence of either a non-silencing siRNA control or p21+p16 siRNA. The efficiency of siRNA transfer was observed by fluorescence microscopy of Cy3-labeled control siRNA. Viability was determined by direct counting of cells before and after electroporation. The ability of p21+p16 siRNA to decrease the protein expression of p21Cip1 and p16INK4a was determined by semi-quantitative analysis of western blots. The effect of siRNA treatment on cell cycle progression and proliferation was determined 1, 5, and 11 days after electroporation by counting Ki67-positive cells and total DAPI-stained nuclei. RESULTS siRNA was efficiently transferred to HCEC by the electroporation method. The average cell loss was 41.25% at 24 h following electroporation. Protein levels of both p21Cip1 and p16INK4a were significantly decreased as the result of p21+p16 siRNA treatment. This treatment significantly increased the average number of Ki67-positive cells over controls and increased the total number of cells in a time-dependent manner. CONCLUSIONS Both p21Cip1 and p16INK4a are involved in negative regulation of the cell cycle in HCEC and, thereby, provide an effective barrier to cell division. The siRNA-induced reduction in expression of these proteins increased the number of cells entering the cell cycle, as well as total cell numbers. Thus, reduction of the levels of p21Cip1 and p16INK4a could be useful in the development of treatments to induce transient cell division to increase corneal endothelial cell density.