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. 2016 Jan 18;9(1):29-32.
doi: 10.18240/ijo.2016.01.05. eCollection 2016.

Effect of NF-κB p65 antisense oligodeoxynucleotide on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2

Chao Liu  1 , Xiao-Li Wu  2 , Xin-Yi Wu  3 , Zhen-Hua Zhang  4 , Xiao-Hua Liu  5
Affiliations

Effect of NF-κB p65 antisense oligodeoxynucleotide on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2

Chao Liu et al. Int J Ophthalmol. .

Abstract

Aim: To study the inhibition of nuclear factor kappa-B p65 (NF-κB p65) antisense oligodeoxynucleotide (ASODN) on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2 (TGF-β2) in vitro.

Methods: NF-κB p65 ASODN and NF-κB p65 missense oligodeoxynucleotide (MSODN) were designed and synthesized. Human lens epithelial cell line (HLE B-3) cells were prepared for study and divided into 7 groups. Control group was HLE B-3 cells cultured in vitro in dulbecco's modified eagle medium (DMEM). T1, T2, and T3 group were HLE B-3 cells cultured in vitro in DMEM with 10 ng/mL TGF-β2 for 6h, 12h, 24h respectively. A+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65 ASODN for 24h. M+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h. The negative control group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after cultured with transfer agent (HiPerFect) for 24h. Cell morphology was observed at different time points using an inverted microscope. The expression of NF-κB p65 mRNA was detected with reverse transcription-polymerase chain reaction (RT-PCR), and the expression of α-smooth muscle actin (α-SMA) protein was assayed with ELISA.

Results: With the TGF-β2 stimulation prolongation, the expression of NF-κB p65 mRNA and α-SMA protein increased in T1, T2, T3 groups compared with the control group, and the difference was statistically significant (P<0.05). NF-κB p65 ASODN lowered the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2. NF-κB p65 MSODN and HiPerFect did not lower the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2. The difference between control group and A+T group was not statistically significant (P>0.05), but the difference among A+T group and other groups was statistically significant (P<0.05).

Conclusion: NF-κB p65 ASODN could lower the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2, and antagonized TGF-β2-induced transdifferentiation of HLE B-3 in vitro. NF-κB p65 ASODN could be used as a new biological therapeutic target of posterior capsular opacification.

Keywords: antisense oligodeoxynucleotide; lens epithelial cells; nuclear factor kappa-B p65; transforming growth factor-β2; α-smooth muscle actin.

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Figures

Figure 1
Figure 1. HLE B-3 cells cultivated with the 10 ng/mL TGF-β2 for 12h.
Figure 2
Figure 2. The agarose gel electrophoresis result of PCR.
See this image and copyright information in PMC

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