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PlGF gene knockdown in human retinal pigment epithelial cells

  • Basic Science
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Abstract

Background

To evaluate the knockdown of placental growth factor (PlGF) gene expression in human retinal pigment epithelium (RPE) cells and its effect on cell proliferation, apoptosis and angiogenic potential of RPE cells.

Methods

Human RPE cells were isolated by dispase I solution and cultured in DMEM/F12 supplemented with 10% fetal calf serum (FCS). A small interfering RNA (siRNA) corresponding to PlGF mRNA and a scrambled siRNA (scRNA) were introduced into the cells. Cell proliferation and cell death were examined by ELISA. PlGF mRNA and protein were quantified by real-time polymerase chain reaction (PCR) and western blot. The levels of gene expression for human retinal pigment epithelium-specific protein 65 kDa (RPE65), cellular retinaldehyde-binding protein (CRALBP) and tyrosinase were examined by real-time PCR. The angiogenic activity of RPE cell-derived conditioned media was assayed by a tube formation assay using human umbilical vein endothelial cells (HUVECs).

Results

At a final siRNA concentration of 20 pmol/ml, the transfection efficiency was about 80%. The amount of PlGF transcripts was reduced to 10% after 36 h of incubation, and the amount of PlGF protein in culture supernatant was significantly decreased. Suppression of PlGF gene had no effect on RPE cell proliferation and survival, and there were no notable changes in the transcript levels of RPE65, CRALBP or tyrosinase for the cultures treated by siRNA cognate to PlGF. Vascular tube formation was efficiently reduced in HUVECs.

Conclusions

Our findings present PlGF as a key modulator of angiogenic potential in RPE cells of the human retina.

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Acknowledgements

This work was supported by National Institute of Genetic Engineering and Biotechnology (NIGEB) through grant no.253. Our gratitude and sincere thanks to Zahra Ataei, Pezhman Fahim, Ali Talebian, Ahmad Gharabaghian, and Tahere Chamani for their contribution in completing this work.

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Authors and Affiliations

  1. Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

    Hassan Akrami & Majid Sadeghizadeh

  2. Department of Biochemistry, National Institute of Genetic Engineering and Biotechnology, P.O. Box: 14965/161, Tehran, Iran

    Zahra-Soheila Soheili & Keynoush Khalooghi

  3. Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Hamid Ahmadieh & Mozhgan Rezaeikanavi

  4. Iranian Blood Transfusion Organization Research Center, Tehran, Iran

    Shahram Samiei

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  1. Hassan Akrami
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  2. Zahra-Soheila Soheili
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  3. Majid Sadeghizadeh
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  4. Hamid Ahmadieh
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  5. Mozhgan Rezaeikanavi
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  7. Keynoush Khalooghi
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Correspondence to Zahra-Soheila Soheili.

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Akrami, H., Soheili, ZS., Sadeghizadeh, M. et al. PlGF gene knockdown in human retinal pigment epithelial cells. Graefes Arch Clin Exp Ophthalmol 249, 537–546 (2011). https://doi.org/10.1007/s00417-010-1567-7

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  • DOI: https://doi.org/10.1007/s00417-010-1567-7

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