title: The polyamidoamine-mediated inhibition of bcl-2 by small hairpin RNA to induce apoptosis in human lens epithelial cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai, China.
authors.forename: Xin-Hua
authors.initials: XH
authors.lastname: Wu
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
authors.forename: Yan-Wen
authors.initials: YW
authors.lastname: Fang
authors.forename: Yong-Xiang
authors.initials: YX
authors.lastname: Jiang
mesh_terms: D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D053148:Caspase 3 / Q000096:biosynthesis; D002460:Cell Line; D045304:Cytochromes c / Q000096:biosynthesis; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry; D011073:Polyamines / Q000737:chemistry*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis*; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection / Q000379:methods
chemical_list: D017304:Annexin A5; D008081:Liposomes; C531249:Poly(amidoamine); D011073:Polyamines; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D045304:Cytochromes c; D053148:Caspase 3