Abstract
Purpose: :
Autophagy plays a critical role in cellular housekeeping in the RPE. MicroRNAs have recently been shown to play a critical role in targeting autophagic proteins and thus regulating the autophagic response in cells. The aim of this study was to investigate whether miR-429, which has been previously reported to be downregulated by stimulation of autophagy in human cancer cell lines, regulates autophagy in the RPE.
Methods: :
ARPE19 cells were reverse transfected with 80nM of miR-429 mimic (Applied Biosystems Inc.) or miR negative control with SiPort NeoFX transfection reagent. 24 and 48 hours after transfection the cells in the presence or absence of bafilomycin A1 (autophagosome and lysosome fusion inhibitor) were harvested to immunoblot for p62 and LC3 lipidation levels (markers of autophagy flux). In parallel, cells expressing tandem tagged RFP-GFP-LC3 plasmid were transfected with the mimic and the number of autophagosomes counted. In order to assess the regulation of starvation-induced autophagy, the above experiments were repeated with 3 hours of starvation prior to harvest. Quantitative RT-PCR and Western blot were used to assess the levels of Unc51-like kinase 2 (ULK-2) and autophagy/beclin-1 regulator 1 (AMBRA1), the in silico predicted autophagy pathway targets (TargetScan) of miR-429, after transfection with microRNA mimics.
Results: :
Western Blot showed that that LC3 lipidation (as calculated by the ratio of LC3 II to LC3 I) significantly decreased while p62 levels increased in the RPE under both basal and starvation conditions when miR-429 mimic was overexpressed for 24 and 48 hours compared to the miR negative control. This result was supported by the observation that the number of autophagosomes showed a significant decline in miR-429 mimic transfected cells when compared to untransfected control or the negative control miR transfected cells. Our previous in silico prediction that ULK-2 and AMBRA1 are putative targets of miR-429, was confirmed by qRT-PCR and Western blot which showed significant decline in the messenger RNA and protein levels of ULK-2 and AMBRA1 in RPE overexpressing miR-429.
Conclusions: :
miR-429 plays a critical role in regulating autophagy in the RPE. Our data suggests that miR-429 acts by downregulating two important autophagic initiation factors, ULK-2 and AMBRA1.
Keywords: retinal pigment epithelium