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. 2010 Jan 26:16:105-12.

Down regulation of the PEDF gene in human lens epithelium cells changed the expression of proteins vimentin and alphaB-crystallin

Jing Yang  1 , Lixia Luo, Xialin Liu, Mark I Rosenblatt, Bo Qu, Yuhua Liu, Yizhi Liu
Affiliations

Down regulation of the PEDF gene in human lens epithelium cells changed the expression of proteins vimentin and alphaB-crystallin

Jing Yang et al. Mol Vis. .

Abstract

Purpose: To study the relationship of pigment epithelium-derived factor (PEDF) expression with the expression of vimentin and alphaB-crystallin by lens epithelial cells.

Methods: Lens epithelial cells adhering to anterior capsules taken from young donor eyes aged from 20 to 35 years were cultured and passaged. We designed small interfering RNA (siRNA) constructs to specifically downregulate the expression of PEDF by these primary lens epithelial cells. Quantitative PCR was used to confirm the downregulation of PEDF RNA expression following infection of lens epithelial cells. To determine whether altering the expression of PEDF would effect the expression of vimentin or alphaB-crystallin, we performed western blotting 48 h after expression of the PEDF-directed siRNA.

Results: PEDF RNA expression in the human lens epithelial cells was strongly downregulated by the three separate siRNA constructs. Western blotting revealed that the downregulation of PEDF expression resulted in a concomitant decrease in expression of vimentin and an increase in alphaB-crystallin protein.

Conclusions: Decreased expression of PEDF in primary human lens epithelial cells resulted in a decrease in the expression of vimentin and the increase of alphaB-crystallin expression, two proteins critical for maintaining lens clarity.

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Figures

Figure 1
Figure 1
Images of human primary lens epithelial cells. Lens epithelial cells adhering to anterior capsule on the dish (A); 5 days later the human lens epithelial cells migrate from the anterior capsule to the dish (B).
Figure 2
Figure 2
Images of human primary lens epithelial cells infected with different lentivirus. A, C, E, G, and I were taken under bright field. B, D, F, H, and J were taken under green fluorescent protein (GFP) field. A and B show the control group; the human primary lens epithelial cells were not infected in this group. C and D show group1 (mock group) infected with shRNA-scramble (random siRNA sequence). E and F show group 2, which is infected with shRNA1. G and H show group 3, which is infected with shRNA2. I and J show group 4, which is infected with shRNA3. The control group (B) has no GFP fluorescence, while almost 80–90% of the cells of group1, 2, 3, and 4 have green fluorescence.
Figure 3
Figure 3
The relative RNA level of pigment epithelium-derived factor (PEDF) in human primary lens epithelial cells after RNA interference. Q-PCR was used to quantify the PEDF mRNA transcript 48 h after the infection. The relative PEDF mRNA expression level was reduced (p<0.01) in the sample 2 (shRNA1), sample 3 (shRNA2), and sample 4 (shRNA3) compared with control group. The mRNA transcripts were not significant differences (p>0.05) between control group and sample 1 (shRNA1 with random RNA sequence). The PEDF mRNA was significantly down regulated with RNAi in group 2, 3, and 4. We used One-way analysis of variance (ANOVA); each experiment was repeated 3 times (n=3).
Figure 4
Figure 4
The relative levels of vimentin and αB-crystallin in Human primary lens epithelial cells after RNA interference (RNAi), measured by western blot. The internal control is Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; A). The expression of vimentin in different groups of human primary epithelial lens cells measured 48 h after lentiviral infection. The levels of Vimentin in the control group are considerably higher compared to shRNA-1, 2, 3 groups (p<0.05). There are no significant differences between the levels of Vimentin detected in the control group and the shRNA-scramble group (p>0.05, shRNA-scr with random RNA sequence). One-way analysis of variance (ANOVA) were applied to each treatment (n=3), and the standard error of the mean (SEM) was used in the graph (B). The expression of αB-crystallin in different groups of human primary epithelial lens cells measured 48 h after lentiviral infection. The levels of αB-crystallin in the control group were considerably lower compared to shRNA-1, 2, 3 groups (p<0.05). There were no significant differences between the levels of αB-crystallin detected in the control group and the shRNA-scramble group (p>0.05, shRNA-scr with random RNA sequence). One-way analysis of variance (ANOVA) was applied to each treatment (n=3),and the standard error of the mean (SEM) was used in the graph (C).
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References

    1. Tombran-Tink J, Shivaram SM, Chader GJ, Johnson LV, Bok D. Expression, secretion, and age-related down regulation of pigment epithelium-derived factor, a serpin withneurotrophic activity. J Neurosci. 1995;15:4992–5003. - PMC - PubMed
    1. Wu YQ, Becerra SP. Proteolytic activity directed toward pigment epithelium-derived factor in vitreous bovine eyes. Implications of proteolytic processing. Invest Ophthalmol Vis Sci. 1996;37:1984–93. - PubMed
    1. Steele FR, Chader GJ, Johnson LV, Tombran-Tink J. Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family. Proc Natl Acad Sci USA. 1993;90:1526–30. - PMC - PubMed
    1. Kuszak JR. Embryology and histology of the lens. Duane’s Clinical Ophthalmology Vol 1. Philadelphia; Lippincot-Raven: 1995. p. 1–9.
    1. Li WC, Kuszak JR, Dunn K, Wang RR, Ma W, Wang GM, Spector A. Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals. J Cell Biol. 1995;130:169–81. - PMC - PubMed

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