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Congxiao Zhang, Kiyoharu J Miyagishima, Ruchi Sharma, Malika Nimmagadda, Genqing Liang, Arvydas Maminishkis, Kapil Bharti, Sheldon S Miller; Hypoxia-induced Alterations of miR-204 Regulates Autophagy Activity in Human Retinal Pigment Epithelium (hRPE). Invest. Ophthalmol. Vis. Sci. 2020;61(7):3110.
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Explore the possibility of hypoxia-induced alterations in miR-204 and RPE autophagic activity as part of a pathogenic response pathway underlying retinal diseases, such as late onset retinal degeneration (L-ORD) or age-related macular degeneration (AMD).
Hypoxic induction of primary human RPE or iPSC - derived RPE was performed by incubating cells in 3% O2 and 5% CO2. For qRT-PCR assay, mRNA or miRNA were quantified by TaqMan TM or SYBR green assay. Housekeeping genes (GAPDH and TBP) or microRNA Let-7a and miR-16 were used as controls. Immunoblots were probed with antibodies against VHL and LC3, using beta-actin or beta-tubulin as loading controls.
Following 6 hours of hypoxia, expression of VHL and miR-204 were decreased in primary human RPE cells. The acute exposure to hypoxia increased conversion of LC3-I to L3-II - a direct target gene of miR-204. Bafilomycin A1, a V-ATPase inhibitor that prevents lysosome acidification and degradation of the autophagosome/LC3-II, did not further increase levels of LC3-II. Under normoxia, the relative expression of miR-204 in iPSC-derived RPE from L-ORD patients were identical to controls. Following hypoxia, the levels of miR-204 were reduced in both patient -derived L-ORD iPSC-RPE cells and their controls, but this reduction was smaller in patient compared to control cells. Although miR-204 - regulated autophagy activity in L-ORD patient-derived RPE is disrupted, its mechanism of activation remains unclear.
In human RPE, miR-204 expression is regulated by acute hypoxia-driven decreases in VHL, similar to findings in clear renal cell carcinoma. miR-204 acts to regulate the homeostatic level of RPE autophagy, suggesting it as an important endogenous modifier of RPE disease pathogenesis following induction of hypoxia and perhaps other cellular metabolic stressors..
This is a 2020 ARVO Annual Meeting abstract.
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