Lens Transcription Factors And Crystallin Expression In Rat Posterior Capsule Opacification

Purpose:To establish a model of posterior capsule opacification (PCO) and examine the expression of transcription factor genes and crystalline genes. To evaluate the feasibility of using short hairpin RNA (shRNA) for selective inhibiting Foxe3 expression in HLEC-B3, and observe the effects of Foxe3 gene silencing on the HLEC-B3 growth.Methods:An extracapsular lens extraction (ECLE) was performed in Spaque-Dawley rats. The animals were killed at 0 hour, 3, 7, 14 days, 1, 2, and 3 months after surgery. The eyes were examined with light microscopy and take picture at various time points. Semi-quantitative reverse transcription PCR (RT-PCR) and immunofluorescence were performed to detect the expression of transcription factors (Pax6, Prox1, and Foxe3). Semi-quantitative RT-PCR was performed to detect the expression ofα-,β-,andγ-crystalline.Biosynthesized double-strand short hairpin RNA (shRNA) targeting Foxe3 gene were transfected into HLEC-B3 with Lipofectamine^TM2000 reagent. The subsequent changes in Foxe3 mRNA level were determined by semi-quantitative reverse transcriptase PCR and real-time PCR after 48h incubation. Changes in Foxe3 protein were determined by Western blot and immunofluorescence after 72h incubation. Flow cytometry, cell growth curve and MTT assay were employed to examine the alterations in cell cycle and cell proliferation.Results:PCO was noted in all rats within 3 days after ECLE. Multilayered lens epithelial cells were present throughout the lens capsule, and lens fibre differentiation occurred in peripheral capsular bag. All eyes showed development of clinically evident PCO and Seomerring's ring at 7 days after ECLE. 1 month after surgery, all capsular bags were filled with new semitransparent lenticular structures, displaying an established equator with well differentiated bow regions.The mRNA-expression quantity ofαA,αB,βB1,βB2,βA2,γD-crystalline, Pax6, and Proxlincreased gradually by semi-quantitative RT-PCR, which were equal to the quantity of the normal lens preoperation; Pax6, Prox1, Foxe3 were all expressed during the formation of PCO. Pax6 is mainly expressed in LEC and newer regenerated lens fibre cells, indicative of an important role for Pax6 gene in determination of the regeneration potential. Prox1 is expressed both in LEC and differentiating lens fibre cells, indicative of a role of fibre differentiation and elongation. Foxe3 is confined to LEC, indicative of a role related with promoting survival and proliferation of LEC.The expression of Foxe3 mRNA was reduced by 60.93% at 48 h, and protein was reduced by 61.54% at 48 h compared with those in blank control treated by shRNA interference. The cell cycle arrest at G0-G1 phase accompanied by a reduction of cell proliferation by 67%, and promoted cell apoptosis. Nonspecific siRNA and other control groups could not affect cell proliferation, Foxe3 mRNA and protein expression in HLEC-B3.Conclusion:1. PCO occured soon after ECLE in SD rat, and this model appears to be valuable for studying the pathogenic mechanisms of PCO after cataract surgery or lens regeneration.2. Lens epithelial ceils proliferation and fiber differentiation are involved in the formation of PCO.3. There is same genetic program operates in both embryonic lens development and PCO, at least partly.4. Foxe3—shRNA interference can effectively inhibit Foxe3 gene expression in LECs and cell proliferation, suggesting that Foxe3—shRNA may be potential to prevent PCO.