miR302a and 122 are deregulated in small extracellular vesicles from ARPE-19 cells cultured with H2O2

Age related macular degeneration (AMD) is a common retina-related disease leading to blindness. Little is known on the origin of the disease, but it is well documented that oxidative stress generated in the retinal pigment epithelium and choroid neovascularization are closely involved. The study of circulating miRNAs is opening new possibilities in terms of diagnosis and therapeutics. miRNAs can travel associated to lipoproteins or inside small Extracellular Vesicles (sEVs). A number of reports indicate a significant deregulation of circulating miRNAs in AMD and experimental approaches, but it is unclear whether sEVs present a significant miRNA cargo. The present work studies miRNA expression changes in sEVs released from ARPE-19 cells under oxidative conditions (i.e. hydrogen peroxide, H2O2). H2O2 increased sEVs release from ARPE-19 cells. Moreover, 218 miRNAs could be detected in control and H2O2 induced-sEVs. Interestingly, only two of them (hsa-miR-302a and hsa-miR-122) were significantly under-expressed in H2O2-induced sEVs. Results herein suggest that the down regulation of miRNAs 302a and 122 might be related with previous studies showing sEVs-induced neovascularization after oxidative challenge in ARPE-19 cells.

Pathway analysis and prediction of miRNA targets regulated by oxidative stress. Subsequently, the role of the under-expressed miRNAs in treated ARPE-19 cells and in sEVs from treated cells (seven in cells and two in sEVs), was analysed. For that reason, two independent ¨in silico¨ analysis were performed in order to determine potential biological processes related to oxidative stress induction. The analysis of the KEGG pathway, regulated by Let-7a, miR-518d-3p, miR-521, miR-338-5p, miR-548b-5p, miR-205, and miR-302c, shows a large number of pathways involving these miRNAs. Among them, cell cycle, adherent junction, p53 signalling pathway, and HIF-1 signalling pathway are the most relevant (Fig. 6A). Both sEV miRNAs, miR-302a and miR-122, are involved in different pathways, such as TGF-beta signalling pathway, FoxO signalling pathway, and cell cycle (Fig. 7A).
Potential targets of miRNAs. In order to identify biological functions of the validated miRNAs and to select their putative targets, two programs were handled: DIANA TOOLS mirPath and Target Scan Human. The outcome on sEV miRNA and Cell miRNA related pathways are not completely equivalent. Most of the significantly involved KEGG are related to cancer or cell cycle pathways (Figs. 6B and 7B).

Discussion
The study of circulating miRNAs on AMD has been addressed to find biological markers which might help implementing an early diagnosis for the disease, or to find new therapeutic strategies 18 . Although neovascular processes and oxidative stress are well known characters involved on AMD, the origin of the disease is far from being completely understood. Several studies were focused in identifying neovascularization-related miRNAs or oxidative www.nature.com/scientificreports www.nature.com/scientificreports/ stress-related ones [23][24][25][26] . On this line, the use of RPE cell cultures, as ARPE-19 under oxidative challenges (eg. H 2 O 2 , rotenone or EtOH), results useful to study the RPE response to oxidative stimuli [27][28][29] .
As mentioned earlier, 600 µM was the highest H 2 O 2 concentration used which increased ROS levels without generating early apoptosis (800 µM H 2 O 2 exposure significantly increased early apoptosis). Other authors have used different H 2 O 2 ranges (200-600 µM) and some differences can be found in terms of cell viability (XTT or MTT), apoptosis and ROS production 7,27,30 . Plausibly, the differences on time exposure (12-24 h) or cell confluence level could explain these discrepancies.
Fitting with previous data, pro-oxidant challenge resulted on significant high sEVs release from ARPE-19 cells. It is well documented that EtOH or high glucose conditions resulted in the same response increasing sEVs release from ARPE-19 cells 28,31 .
Extracellular or circulating miRNAs can be included on sEVs, associated to lipoproteins or proteins 13 . In this respect, we have just focused on miRNAs contained as sEVs cargo (sEV miRNA). In spite of the fact that in sEVs only 218 out of 384 miRNAs could be detected, only two sEV miRNAs were significantly high in control released sEVs than in sEVs from treated cells. The hsa-miR-302a and hsa-miR-122 have already been seen in sEVs confirming our results [32][33][34][35] .  www.nature.com/scientificreports www.nature.com/scientificreports/ When comparing the two sEV miRNAs, hsa-miR-302a and hsa-miR-122, to those miRNAs on ARPE-19 cells (Cell miRNA), no matches could be found. Surprisingly, the sEV miRNA hsa-miR-302a and the Cell miRNA hsa-miR-302c belong to the miR 302/367 La-related protein 7 (LARP7) intragenic cluster. This includes hsa-miR-367, hsa-miR-302d, hsa-miR-302a, hsa-miR-302c, and hsa-miR-302b. This cluster is involved in several processes coordinating proliferation, differentiation, pluripotency maintenance, and cell reprogramming 36 . Moreover, the cluster, regulates TGF pathway, PI3K-AKT and BMP cell signaling [37][38][39] . Additionally, hsa-miR-302a acts as a tumor suppressor 40 and repressor of cell division, and more concretely, VEGFA is one of the direct targets for this miRNA 41 . In addition, hsa-miR-122 has been related to VEGFC 42 . Interestingly, low hsa-miR-302a/hsa-miR-122 expression levels are inversely related to VEGF levels in hepatocellular carcinoma, promoting vascular changes 43,44 . In addition, miR-122 seems to have a role against oxidative stress, since the use of pre-miR-122 protects from H 2 O 2-induced oxidative stress 45 , targeting the mitochondrial ribosomal protein S11 46 .
Recent data from other groups indicate how diverse oxidative insults -EtOH or high glucose, and now H 2 O 2lead to increased sEVs release from ARPE-19 cells 28,31 . Furthermore, those oxidative-induced sEVs were capable of promoting neovascular processes in endothelial cell cultures, whereas control-released sEVs inhibited this phenomenon 28,31 . In view of these findings, we hypothesize that sEVs hsa-miR-122 and hsa-miR-302a decreased levels could play a role on angiogenesis involving oxidative stress related pathways. Several reports on AMD or experimental AMD, have reported significant changes on miRNAs (see Table 2). Among them, hsa-miR-23a is present in five reports 7,16,17,42,47 . This miRNA seems to be downregulated in both AMD patients and H 2 O 2 -treated ARPE-19 cells. In fact, H 2 O 2 induced-apoptotic cell death is significantly observed in ARPE-19 cells after hsa-miR-23a inhibition 7 . Fitting with this, we found significant expression differences on hsa-miR-23a after H 2 O 2 exposure (FC = 0.79). Table 2 summarizes those Cell miRNAs matching with previous reports on AMD or AMD experimental models. Besides, there are reports that locate the sEVS-miRNAs in eye tissue. MiR-302 expression was observed already in RPE cells by Li and collaborators, playing an important role in the RPE differentiation 48 . Other authors observed a miR-122 differential expression on canine retinas 49 , in aqueous humor 50 and are related in fact with diabetic retinopathy 51 . In agreement with others, up/down regulated miRNAs are related to several and different cell signaling pathways. After seeing the results in both sEV miRNA and Cell miRNAs related pathways, cancer related pathways are commonly involved in many of the deregulated miRNAs. More research must be performed on these generic pathways to determine the concrete role of these cell signaling routes.

Material and Methods
Cell culture. Arising retinal pigment epithelium (ARPE-19) human cell line was obtained from American Type Culture Collection (ATCC, Barcelona, Spain) at passage 19. ARPE-19 cells were cultured in Dulbecco's modified Eagle's DMEM/F12 (Invitrogen, Carlsbad, CA, USA), as previously described 21 . Cells were used until passage 30. Cells were cultured to 80-90% confluence at a starting density of 1 × 10 6 cells/cm 2 in different plates depending on the technique. After 2 days, the cells were treated for 24 h with 600 µM H 2 O 2 (Scharlau, Senmenat, Spain), using filtered media with 1% of Fetal Bovine Serum, exosome-depleted (FBS; Thermo Fisher Scientific, Gibco, USA). Cells and supernatant were collected and preserved for future experiments.
Determination of intracellular ROS. Intracellular ROS levels were measured using dihydroethidium, (DHE; Thermo Fisher Scientific, Waltham, MA, USA), which is a superoxide indicator. This molecule has a blue fluorescence, but, when oxidized to ethidium, it stains DNA in red. ARPE-19 cells were seeded at 6 × 10 3 cells/ well in a 96 well plate. Cells were rinsed with PBS (phosphate-buffered saline) twice and incubated with 5 μM of DHE during 30 min at 37 °C and 5% CO 2 . ROS levels were measured by a fluorescence multiplate reader (Victor X5; Perkin Elmer) excited at 518 nm and read at 605 nm. sEVs isolation and size distribution. sEVs isolation was performed by successive ultracentrifugation as previously reported 21 . The sEVs pellet was stored at 4 °C until further processing in PBS solution. For microarray assay, sEVs were isolated using ExoQuick-TC (Systems Biosciences, Mountain View, CA, USA) following the manufacturer's instructions. sEVs identity was confirmed by the nanoparticle tracking system NanoSight NS300 following manufacturer's protocols (Malvern Instruments, Malvern, UK).

Apoptosis detection.
Electron microscopy. sEVs pellets were resuspended in PBS and ultracentrifuged at 120.000 × g for 70 min at 4 °C. After that, approximately 10 µg of the samples were resuspended in PBS on parafilm. The sample was fixed by depositing a drop of 2% Paraformaldehyde on the parafilm and placing the grid (Mesh with Formvar) on top of the drop. Negative staining was performed with 2% uranyl acetate. Photomicrographs were obtained using the transmission electron microscope FEI Tecnai G2 Spirit (FEI Europe, Eindhoven, Netherlands) using a digital camera Morada (Olympus Soft Image Solutions GmbH, Münster, Germany). EVs were identified under the microscope solely based on size and morphology.

RNA isolation and miRNA expression analysis.
To perform microarray analysis, ARPE-19 cells from 4 separate cultures were exposed to control and H 2 O 2 600 µM treatment for 24 h. Total RNA was extracted using SeraMir Kit (System Biosciences, Mountain View, CA, USA) according to manufacturer's instructions. Therefore, four microarrays were performed for each condition: control, ARPE-19 cells exposed to H 2 O 2 600 µM, sEVs released by control cells and sEVs released by ARPE-19 cells exposed to H 2 O 2 600 µM.

Array analysis. We analyzed miRNA expression differences between ARPE-19 control cells and ARPE-19
treated with H 2 O 2 . Moreover, differences in miRNA expression between EVs released by ARPE-19 cells treated and EVs released by ARPE-19 control cells were also studied. Differences were analyzed using a t-test study from genefilter package from R Bioconductor. P-values were adjusted by the Benjamini-Hochberg method. MiRNAs that presented an adjusted p-value < 0.05 were considered statistically significant. Significantly modified miRNAs from different samples were represented in a hierarchical clustering heatmap representation. Heatmaps were performed using heatmap.3 package from R Bioconductor.
Analysis of miRNA target genes. In silico analysis of the pathways in which the miRNAs regulated by H 2 O 2 were involved using DIANA TOOLS mirPath v.3 algorithm (http://snf-515788.vm.okeanos.grnet.gr/). Moreover, we carry out an analyse of the putative miRNAs target using TargetScanHuman (http://www.targetscan.org/vert_72/). Quantitative real-time PCR validation. Quantitative real time PCR (qRT-PCR) was used to validate the miRNA expression profile of the selected miRNAs in an independent sample set. The RNA was isolated from ARPE-19 cells by miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.