Purpose: To investigate whether apoptosis of human lens epithelial cells (HLECs) can be induced with the polyamidoamine (PAMAM)-mediated inhibition of bcl-2 (b-cell lymphoma 2) by small hairpin RNA (shRNA).
Methods: HLECs (SRA01/04) were transfected with the fifth generation of PAMAM (PAMAM G5) by bcl-2 shRNA. At 24, 48, and 72 h after transfection, the transfection rate was measured by flow cytometry. The transfection rates mediated by PAMAM and liposome were compared. The bcl-2 mRNA level was detected by real-time PCR. Whole cell protein was extracted and the bcl-2 protein level was detected by western blotting. The percentage of HLECs undergoing apoptosis was measured by Annexin V-FITC/PI staining. The nuclear morphology of HLECs was observed by staining with Hoechst 33258. The expression of cytochrome c and the activity of cleaved caspase-3 were analyzed by western blotting.
Results: At 24, 48, and 72 h after transfection, the rate of transfection of bcl-2 shRNA mediated by PAMAM was higher than in the liposome-mediated group (p<0.05). The mRNA and protein levels of bcl-2 were greatly downregulated. The percentage of HLECs undergoing apoptosis was greatly improved. Hoechst staining showed that bcl-2 shRNA transfected cells had a lower growth status with nuclear fragmentation. The expression of cytochrome c and the activity of cleaved caspase-3 was greatly improved (p<0.05).
Conclusions: PAMAM-mediated bcl-2 shRNA can downregulate the expression of bcl-2 and induce the apoptosis of HLECs by engaging the mitochondrial pathway, including catalytic activation of the caspases.
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