Purpose: The retina is being exposed to a variety of environmental stress, including light-induced damage, oxidative stress and inherited mutations that can lead to protein misfolding. Accumulation of unfolded proteins in ER lumen leads to ER stress. In response to ER stress, synthesis of total proteins is decreased while chaperons expression such as GRP78 is elevated. Sustained endoplasmic reticulum stress in RPE cells results in apoptosis and retinal diseases such as AMD and RP. microRNAs as endogenous regulators play critical role in modulating gene expression. Previous studies suggest that miR-30d, miR-181a and miR-199a-5p down regulate GRP78 expression by direct targeting its 3′UTR.To search for the expression of miR-30d, miR-181a and miR-199a-5p, as principal players in GRP78 modulation, and to evaluate GRP78 measure in human newborn and adult RPE cells in culture.

Methods: Adult and fetal human RPE cells were isolated, characterized and cultured according to standard protocols. Total RNA was extracted by Tripure isolation reagent and treated with DNase for eliminating genomic DNA. cDNA was synthesized by stem-loop primers specifically designed for each miR-30d/181a/199a-5p and then applied to perform stem-loop RT-qPCR method for measuring individual miRNAs. GRP78 expression was also evaluated in extracted RNAs using specific, conventional primers for GRP78. RPE cells in culture were transfected by the constructs containing GRP78 using calcium phosphate precipitation method. The aforesaid sample used as positive controls for GRP78. After 48 hours cells’ lysate was prepared using RIPA lysis buffer reagent and applied to perform western blot for GRP78. Protein extraction was also done for RPE cells isolated from donors at different ages and GRP78 expression was evaluated.

Results: We are reporting that miR-30d/181a/199a-5p in both of the adult and fetal human RPE cells, are expressed. Moreover our results demonstrated that GRP78 was under detection limit in isolated RPE cells from both of the adult and newborn human globes.

Conclusion: Our data showed that detectable expression of miR-30d/181a/199a-5p in hRPE cells is concomitant with neglect able amount of GRP78 in both of the adult and fetal human RPE cells in culture.