[Inhibition of bcl-2 by shRNA to induce apoptosis in human lens epithelial cells].
OBJECTIVE
To investigate whether apoptosis of human lens epithelial cells (HLEC) can be induced by inhibition of bcl-2 shRNA.


METHODS
It was an experimental study. Two pairs of oligonucleotides were synthesized and inserted into plasmid PGCsi to generate shRNA eukaryotic expression vectors, named P1 and P2. At 48 hours after transfection in HLEC (SRA01/04) with Lipofectamine 2000, the whole cell protein was extracted and detected by Western blot. The bcl-2 mRNA level was detected by real-time PCR. The percentage of HLECs undergoing apoptosis was measured by Annexin V-PI staining. The activity of caspase-3 was analyzed by Western blotting.


RESULTS
The shRNA eukaryotic expression vectors were constructed successfully. At 48 hours after transfection, the rate of transfection of P1 and P2 was about 44.1% and 47.2% respectively. The protein and mRNA level of bcl-2 was 0.435 and 0.476, greatly downregulated (F = 1672.4, P < 0.05). The percentage of HLEC undergoing apoptosis was 42.3% and 45.4%, greatly improved (F = 1756.2, P < 0.05). The activity of caspase-3 was greatly improved.


CONCLUSIONS
P1 and P2 can both down-regulate the expression of bcl-2, and induce the apoptosis of HLEC.
Wu
Xin-Hua
XH
Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai 200031, China.
Lu
Yi
Y
Fang
Yan-Wen
YW
Jiang
Yong-Xiang
YX
Tian
Jie
J
D017209:Apoptosis; D002386:Cataract; D002460:Cell Line; D015536:Down-Regulation; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D019254:Genes, bcl-2; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D014162:Transfection
D034741:RNA, Small Interfering