The proliferative inhibition and apoptosis promotion of Smac on human lens epithelial cells

Background

Posterior capsular opacification (PCO) is a primary complication after extracapsular cataract extraction.The mechanism of PCO is associated with proliferation, migration and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs). To explore the target treatment of PCO is very important.

Objective

This study was to investigate the biological effects of second mitochondria-derived activator of caspases (Smac) on the proliferation and apoptosis of LECs.

Methods

Human LECs line (HLE-B₃) and Smac-overexpressed LECs line were cultured, and the cells were transfected using small interfering RNA (siRNA)-Smac3 plasmid with green fluorescent protein (GFP) for 24 hours.Different concentration of transforming growth factor -β₂ (TGF-β₂) (5, 10, 20 and 50 μg/ml) or 200 μmol/L H₂O₂ were added respectively into the culture medium to establish PCO model and oxidative stress model.Cell counting kit-8 (CCK-8) assay was used to compare the cell proliferative activity among PBS group, TGF-β₂ group and Smac-hyperexpression+ TGF-β₂ group.Flow cytometry was used to evaluate the apoptotic rate of the PBS group, H₂O₂ group and siRNA-Smac+ H₂O₂ group.The expressions of Smac, caspase-3 and proliferating cell nuclear antigen (PCNA) mRNA and their proteins in the cells were detected by real-time quantitative PCR (RT-PCR) and Western blot.

Results

The GFP⁺ cells were≥ 80% 12 hours after siRNA-Smac3 transfection, with the optimal plasmid of siRNA-Smac3.GFP⁺ cell rate was (72.32±2.31)% in the siRNA-Smac3 transfection group, which was significantly higher than that in the blank plasmid group([4.91±0.24]%) (t=116.342, P<0.001). The relevant expression levels of Smac was 35.21±4.11 in the Smac-hyperexpression group, and that in the blank plasmid group was 15.24±2.48, with a significant difference between them (t=215.47, P<0.05). The cell viability of 20 ng/ml TGF-β₂ affected PBS group, TGF-β₂ group and Smac-hyperepression+ TGF-β₂ group was (98.4±1.7)%, (98.9±0.1)% and (64.2±3.1)%, and the cell viability of Smac-hyperepression+ TGF-β₂ group was significantly lower in the Smac-hyperepression+ TGF-β₂group than that in the TGF-β₂ group (P<0.05). The apoptotic rate in the PBS group, H₂O₂ group and siRNA-Smac+ H₂O₂ group were (2.9±1.2) %, (45.1±4.5)% and (27.5±1.8)%, and the apoptotic rate was evidently lower in the siRNA-Smac+ H₂O₂ group than that in the H₂O₂ group (P<0.05). RT-PCR results showed that the expression levels of caspase-3 mRNA in PBS group, H₂O₂ group and siRNA-Smac+ H₂O₂ group were 0.321±0.103, 0.715±0.112 and 0.479±0.209, respectively.Compared with the H₂O₂ group, the relative expression level of caspase-3 mRNA in siRNA-Smac+ H₂O₂ group was significantly decreased, the difference was statistically significant (P<0.05). The PCNA mRNA expression levels in PBS group, TGF-β₂ group and Smac-hyperepression+ TGF-β₂ group were 0.299±0.013, 0.645±0.102 and 0.490±0.209, respectively.Western blot results showed that the relative expression of caspase-3 protein in siRNA-Smac+ H₂O₂ group and H₂O₂ group was 0.712±0.012 and 0.973±0.051, with significant difference between the two groups (t=132.52, P<0.05). The relative expression of PCNA protein in Smac-hyperepression+ TGF-β₂ group was 0.782±0.212, which was lower than 1.126±0.251 in the TGF-β₂ group (P<0.05).

Conclusions

Smac may prevent and treat PCO by inhibiting the proliferation and promoting apoptosis of human LECs.