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GRP78 knockdown in retinal pigment epithelium (RPE) cells and its effects on inflammatory factors expression

Session Details

Session Title: New drug treatment and technology

Session Date/Time: Friday 27/09/2013 | 14:30-16:00

Paper Time: 15:34

Venue: Hall 3 (Level 0)

First Author: S.Samiee IRAN

Co Author(s):    S. Mohammadi   Z. Soheili   A. Deezagi   H. Ahmadieh     

Abstract Details

Purpose:

Endoplasmic reticulum(ER) plays critical role in protein folding through its chaperons such as glucose regulated protein 78KD (GRP78). Unfolded proteins accumulation in ER causes ER stress process, which reduces total protein synthesize and increases expression of chaperons like GRP78, in response. In adult RPE cells prolongation of ER stress causes apoptosis and retinal degeneration so retinal diseases like Age-related macular degeneration (AMD) and retinitis pigmentosa (RP) will be appeared. The purpose of this study was GRP78 knockdown in RPE cells and investigation of its effects on specific inflammatory factors.

Setting:

National Institute of Genetic Engineering and Biotechnology

Methods:

Adult RPE cells were isolated from human globes, characterized by RPE65 and cytokeratin 8/18 as their specific markers and cultured in Dulbecco's Modified Eagle's Medium (DMEM) /F12 supplement 20% fetal bovine serum (FBS). Propagated cultures were consecutively sub-cultured in DMEM/F12 supplement 10%FBS and between passages 2-5 used in subsequent experiments. Cultures were transfected by GRP78 cognate siRNA to knockdown its expression. Scrambled siRNA treated cells, transfection reagent treated cultures and un-transfected RPE cells were used as controls. The rate of cell proliferation and cell death was assessed by ELISA assay.

Results:

Cell death and cell proliferation assay showed that RNAi treatments had no unwanted effect on apoptotic index and cell proliferation in transfected cells in respect to controls. Real Time PCR showed GRP78 expression was knocked down, caspase 4 expressions was decreased and CRP expression was increased. The amount of transcripts for MCP1, CFH, C5, C3, CD59, and CD55 remained unchanged.

Conclusions:

According to these observations, it appears that GRP78 knock down had no undesired effect on cell death and cell proliferation potential of RPE cells in cultures. It concluded that the amount of caspase4 transcripts is closely related to GRP78 expression. Since caspase 4 is one of the effective dealers in apoptosis, it can be concluded that GRP78 decline can prevent apoptosis. On the other hand increased amount of CRP promotes the clearance of dead cells and boosts anti-inflammatory responses.

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