miR-184 regulates ezrin, LAMP-1 expression, affects phagocytosis in human retinal pigment epithelium...

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/febs.13066

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MiR-184 Regulates Ezrin, LAMP-1 Expression, Affects Phagocytosis in Human Retinal Pigment

Epithelium and is Down-Regulated in Age-related Macular Degeneration

Najiba Murad3, Maria Kokkinaki1, Nishantha Gunawardena3, Mia S. Gunawan3, Yetrib Hathout5, Karolina

J. Janczura4, Alexander C. Theos4, and Nady Golestaneh1, 2, 3

1Department of Ophthalmology

2Department of Neurology

3Department of Biochemistry and Molecular & Cellular Biology

4 Department of Human Science

Georgetown University Medical Center, Washington, DC 20057

5 Center for Genetic Medicine Research (CGMR), Children's National Medical Center, Washington, DC

20010

Running title: miR-184 regulates expression of Ezrin and LAMP-1 and phagocytosis in RPE

Article type : Original Article

Correspondence should be addressed to:

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Dr. Nady Golestaneh, PhD

Departments of Ophthalmology, Neurology,

Biochemistry and Molecular & Cellular Biology

Georgetown University Medical Center

3900 Reservoir Road NW,

Medical-Dental Building, Room NE203

Washington, DC 20057

Phone: 202-687-4309

Email: [email protected]

Key words: miR-184, RPE, Ezrin, LAMP-1, AMD

The authors declare no conflict of interest.

ABSTRACT

MicroRNA 184 (miR-184) is known to play a key role in neurological development and apoptosis

and is highly expressed in mouse brain, mouse corneal epithelium, zebrafish lens, and human retinal

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pigment epithelium (RPE). However, the role of miR-184 in RPE is largely unknown. We investigated

the role of miR-184 in RPE and its possible implication in age-related macular degeneration (AMD).

Proteomic analysis identified the Ezrin (EZR) gene as a target of miR-184 in human RPE. EZR is a

membrane cytoskeleton cross-linker that is also known to bind to Lysosomal-Associated Membrane

Protein 1 (LAMP-1) during the formation of phagocytic vacuoles. In ARPE-19 cells, inhibition of miR-184

resulted in up-regulation of EZR mRNA and EZR protein, and induced down-regulation of LAMP-1. The

inhibition of miR-184 decreased EZR-bound LAMP-1 protein levels and affected phagocytic activity in

ARPE19 cells. In primary culture of human RPE isolated from eyes of AMD donors (AMD RPE), miR-184

was significantly down-regulated compared to control (normal) RPE. Down-regulation of miR-184 was

consistent with significantly lower levels of LAMP-1 protein in AMD RPE, and overexpression of MIR-184

in AMD RPE was able to rescue LAMP-1 protein expression to normal levels. All together, these

observations suggest a novel role for miR-184 in RPE health and support a model proposing that down-

regulation of miR-184 expression during aging may result in dysregulation of RPE function, contributing

to retinal degeneration.

INTRODUCTION

miR-184 is a highly evolutionarily conserved short non-coding RNA molecule that post-

transcriptionally regulates protein translation and the function of other miRNAs. The location of the

MIR-184 gene has been mapped to the region 25.1 on the q-arm of chromosome 15 [1]. The expression

pattern of MIR184 gene and maturation of the final miR-184 is highly regulated and is tissue- and

developmental stage- specific [2, 3]. The repression of the MIR-184 gene is partly carried out by DNA

methylation factors such as Methyl-CpG binding protein 1 (MBD1) [4]. While primary transcripts and

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precursors of miR-184 have been detected in many tissue types [1, 2, 5-7], mature miR-184 is only highly

enriched in mouse brain [8], suprabasal cells of the mouse corneal epithelium [9], and zebrafish lens,

where it is known to target mediators of neurological development, apoptosis, and cell differentiation

[9, 10]. Furthermore, miR-184 has been implicated in germline development of Drosophila

melanogaster [11].

The retinal pigment epithelium (RPE) is a monolayer of pigmented and polarized cells; the apical

site of RPE is in close proximity to the outer segments of photoreceptors that are phagocytosed [12],

and the basolateral membrane is opposed to Bruch’s membrane and the choroid blood supply [13]. The

RPE plays an important role in retinal homeostasis, including formation of the blood/retina barrier [14,

15], light absorption [16, 17], isomerization of retinol in the visual cycle [18, 19], transportation of

nutrients such as glucose, retinol and fatty acids from blood to the photoreceptors [19, 20],

transportation of ions and water from the subretinal space to the blood [21], establishment of the

immune privilege of the eye [22], and secretion of growth factors [23, 24].

Ezrin (EZR) is a member of the Ezrin, Radixin, Moesin (ERM) proteins family that are reported to

act as linkers between the cytoskeleton and plasma membrane [25]. Ezrin plays a role in the regulation

of cellular adhesion, movement and morphology in epithelia [26] and functions as a protein-tyrosine

kinase substrate in microvilli [25, 27]. Alterations in the expression of Ezrin and other members of ERM

family have also been observed in several types of cancer including breast cancer [28], osteosarcoma

[29] and brain tumors [30]. EZR protein functions by directly binding to proteins including LAMP-1

(Lysosomal-Associated Membrane Protein 1, also known as CD107a) [31], CD44 [32] and the actin

cytoskeleton [33]. The molecular interaction of EZR with CD44 and LAMP-1 is required for metastasis

[31], the formation of phagocytic vacuoles [34], and vesicular sorting [31, 35]. Several miRNAs have

been confirmed to regulate EZR. For example, miR-183 down-regulates EZR expression in lung cancer

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cells [36]. Meanwhile, miR-589, miR-4778, and miR-548c are predicted by bioinformatics analysis to

bind to EZR 3’UTR (http://mirdb.org/miRDB/). LAMP-1 is a membrane glycoprotein that is a key

structural component of the lysosomal membrane along with LAMP-2 [37]. LAMP-1 has been shown to

be involved in phagocytic digestion of photoreceptor outer segments (POS) by RPE, similar to its role in

lysosomal digestion in other cell types [38]. Deficiencies in both LAMP-1 and LAMP-2 are associated

with an increased accumulation of autophagic vacuoles in mouse embryonic fibroblasts [35].

Human RPE exhibit a cell-type specific tandem expression of mature miR-184 [39], however, the

role of miR-184 in RPE is poorly understood. In this study, we begin to explore the role of miR-184 in

human RPE and demonstrate a novel role for miR-184 in the pathophysiology of AMD.

RESULTS

miR-184 down-regulates Ezrin in human RPE

miR-184 is highly evolutionarily conserved among many species from insects to humans (Figure

1A). In order to determine the genes and proteins that are regulated by miR-184 in human RPE, we

performed proteomic analysis after transfecting cells in primary culture with miR-184 inhibitor for 72

hrs. Proteomics, as explained in the method section, revealed a large list of proteins that were either

up- or down-regulated when miR-184 was inhibited. Among these proteins, EZR showed the highest

increase, by 2.12-fold (Figure 1B). Since EZR is shown to promote actin assembly at the phagosome

membrane and to regulate phago-lysosomal fusion [33], we focused on the role of miR-184 in regulating

EZR, and its downstream biological effect on human RPE.

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As shown in Figure 1C by qRT-PCR, MIR-184 is expressed in human primary RPE culture and

ARPE19, but not in HeLa cells. Therefore, we used HeLa cells to study the ectopic overexpression of

miR-184 by transfection. To study the possible interaction of EZR with miR-184, we transfected HeLa

cells with Luciferase-EZR-3’UTR plasmid construct, MIR-184 plasmid construct, or with both Luciferase-

EZR-3’UTR and MIR-184 plasmid constructs for 48 hrs followed by bioluminescence assay. Our data

revealed that the HeLa cells transfected with Luciferase-EZR-3’UTR constructs showed increase in

luciferase activity, whereas this activity was significantly inhibited (P < 0.01) when HeLa cells were

transfected with both Luciferase-EZR-3’UTR and MIR-184 plasmid constructs, suggesting a direct

interaction between 3’UTR of EZR and miR-184 resulting in down-regulation of EZR expression (Figure

2A). To further confirm the inhibitory effect of miR-184 on EZR, we transfected ARPE19 cells with

Luciferase-EZR-3’UTR construct alone, or with both Luciferase-EZR-3’UTR and miRNA-184 inhibitor

followed by bioluminescence assay. Our data showed that ectopic inhibition of miR-184 significantly

increased EZR expression in ARPE19 compared to EZR expression in ARPE19 transfected with Luciferase-

EZR-3’UTR construct alone or in the presence of endogenous miR-184 only (P < 0.01) (Figure 2B). The

interaction of miR-184 and the EZR-mRNA 3’UTR is further supported by the existence of a putative

binding site sequence for the miR-184 seed sequence (GGACGGA) from nucleotides 521 to 525 in the

EZR-3’UTR (Figure 2C).

miRNA-184 affects phagocytosis and the expression of EZR and LAMP-1 proteins in human RPE

Recent studies have shown an important role for EZR in regulating phagosome maturation and

phago-lysosomal fusion therefore regulating phagocytosis [33, 40]. Other studies have reported that

EZR can promote morphogenesis of apical microvilli and basal infoldings in RPE, and is required for

microvilli formation in mouse RPE [41, 42]. To investigate the functional role of miR-184 in regulating

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EZR-dependent phagocytosis in RPE, ARPE19 cells were cultured for two weeks post confluency and

transfected with miR-184 inhibitor or scrambled siRNA for 48h with lipofectamine. To verify the

silencing of MIR-184, we assayed the expression of MIR-184 by qRT-PCR and show that expression was

knocked down by approximately 90% compared to the mock transfection (n=3) (Figure 3A).

Phagocytosis was analyzed using bovine photoreceptor outer segments (POS) by immunostaining using

antibodies to Rhodopsin to verify POS uptake. Analysis of phagocytosis revealed that inhibition of miR-

184 moderately reduced POS uptake in ARPE19 compared to the cells transfected with control siRNA in

a statistically significant manner (P-value < 0.05) (Figures 3B-3D). The moderate but statistically

significant reduction in POS uptake in vitro in ARPE19 transfected with miR-184 inhibitor could be

explained by the transient nature of miR-184 inhibition and the presence of untransfected cells in the

culture.

To verify the effect of miR-184 on genes regulating phagocytosis, we transfected ARPE19 cells

with miR-184 inhibitor and analyzed the mRNA expression of these genes by RT-PCR and qRT-PCR.

Since one study in mouse epithelial cells has shown a competitive RNA network involving miR-184 and

miR-204 [43], we also used miR-204 inhibitor, in parallel, to investigate the possibility of inhibition of

EZR by miR-204. In order to verify the silencing of the MIR-184 gene by its inhibitor, we assayed the

expression of MIR-184 and MIR-204 by qRT-PCR and showed that neither is detectable 72 h after

transfection by electroporation (n=3). We did observe an up-regulation of MIR-184 (1.64 fold) when

miR-204 is inhibited (P = 0.02) (Figure 4A). This suggests that miR-204 might have a regulatory effect on

miR-184. Our data in Figures 4B-4D show that miR-184 regulates EZR and LAMP-1, but not TYRO3 and

AXL mRNAs. EZR mRNA was significantly up-regulated (P < 0.01), while LAMP-1 mRNA was significantly

down-regulated (P < 0.01) in ARPE19 transfected with miR-184 inhibitor compared to cells transfected

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with the control siRNA (Figure 4C, 4D). MiR-204 inhibition had no significant impact on the expression

of LAMP-1, AXL or TYRO3, but significantly increased the levels of EZR mRNA (Figure 4C, 4D) suggesting a

common regulatory role of miR-184 and miR-204 in EZR gene expression. Western blot analysis of the

ARPE19 cells transfected with miR-184 inhibitor for 72 h confirmed the up-regulation of EZR protein and

the down-regulation of LAMP-1 protein in cells transfected with miR-184 inhibitor compared to the

control cells (Figure 4E). However, TYRO3 protein expression levels did not change (Figure 4E).

Expression of LAMP-1 and EZR proteins in Figure 4E was quantified by densitometry analysis, showing

the down-regulation of LAMP-1 protein and up-regulation of EZR protein (Figure 4F).

A recent study revealed the direct molecular interaction of EZR with LAMP-1 in human

melanoma cells [31]. To assess the interaction of EZR and LAMP-1 and the role of miR-184 in affecting

their interaction in human RPE, we transfected ARPE19 cells with miR-184 inhibitor or scrambled siRNA,

and performed co-immunostaining of ARPE19 cells with LAMP-1 and EZR antibodies. Our data show

that EZR and LAMP-1 partially co-localize and that miR-184 inhibitor down-regulates LAMP-1 protein

expression, thus affecting its co-localization with EZR (Figures 5A-5F). Since EZR is shown to be apically

localized in the epithelial cells [44], and LAMP-1, the lysosomal membrane protein, is distributed

throughout the cytoplasm, a partial co-localization of EZR and LAMP-1 in RPE cell culture is expected, as

observed in our Figure 5C-5D. To confirm the interaction between EZR and LAMP-1, we performed

immunoprecipitation of EZR followed by immunoblotting with LAMP-1 antibody. Our data in Figure 5G

show that EZR and LAMP-1 interact and their interaction is down-regulated in ARPE19 transfected with

miR-184 inhibitor. This is corroborated with our findings showing the down-regulation of total LAMP-1

protein expression by miR-184 inhibitor (Figures 5A-5B, 5G) and therefore its lower availability to

interact with EZR. Densitometry analysis of the co-immunoprecipitation experiment further shows the

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down-regulation of interaction between EZR and LAMP-1 by the miR-184 inhibitor (Figure 5H). Together

our data suggests that miR-184 regulates the expression of EZR and its down-regulation inhibits LAMP-1

expression, pivotal proteins involved in phagocytosis and phago-lysosomal fusion.

miR-184 is down-regulated in AMD

Recent studies have shown critical roles for miRNAs in a variety of diseases [45]. Two

independent studies have reported on the regulation of angiogenesis and choroidal neovascularization

by miRNAs [46, 47]. miRNAs may also serve as potential therapeutic targets for AMD [48]. To

investigate the possible involvement of miR-184 in the pathophysiology of AMD, we performed qRT-PCR

to detect the MIR-184 levels in human primary RPE cultures from 5 AMD and 5 normal donors (Table 1).

Our data shows that miR-184 is significantly inhibited in RPE from AMD donors, and in two AMD samples

(AMD 9 and 14), the MIR-184 expression was undetectable by qRT-PCR (Figure 6A). However, the

expression level of MIR-204, the competitor of MIR-184, was not affected in AMD samples (Figure 6A).

In addition, the expression of LAMP-1 was down-regulated in four out of five AMD samples (Figure 6A).

We observed a direct correlation between the lowest expression levels of MIR-184 and inhibition of

LAMP-1 mRNA levels in the AMD samples (Figure 6A, AMD 9 and 14). In accordance with our data

obtained in ARPE-19 with the miR-184 inhibitor (Figures 4-5), these observations further support a

correlation between miR-184 and LAMP-1 expression and suggest that low levels of miR-184 can still

maintain LAMP-1 expression in human RPE (Figure 6A, AMD 19 and 32). Moreover, low expression levels

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of MIR-184 and LAMP-1 genes in the AMD RPE compared to normal RPE suggest an important role for

miR-184 in the disease mechanisms of AMD.

Since we observed a 2-fold increase in the protein levels of EZR when miR-184 was inhibited in

ARPE19 (Figures 4E-4F), we attempted to demonstrate a difference in the protein levels of EZR between

control and AMD primary RPE cultures with low levels of miR-184. However, the Western blot analysis

could not detect such a subtle change (data not shown). Taking into consideration that miR-184 in the

primary AMD RPE is down-regulated but not totally silenced, the changes in Ezrin protein levels, if any,

should be less than 2-fold and therefore hard to detect by Western blot.

To assess whether restoring the miR-184 levels in AMD RPE could rescue the LAMP-1 expression

levels, we selected the two AMD RPE cells with the lowest MIR-184 and LAMP-1 expression and

performed transfection with the MIR-184 overexpressing plasmid construct followed by qRT-PCR and

Western blot analysis. Our data in Figures 6B and 6C show that transfection with MIR-184

overexpressing plasmid for 72 hours restored the levels of MIR-184 in the AMD RPE (Figure 6B) and

therefore rescued the LAMP-1 mRNA levels in the transfected cells compared to AMD RPE cells

transfected with an empty vector as a control (Figure 6C). Western blot analysis of the AMD RPE cells

transfected with MIR-184 plasmid confirmed the rescue of LAMP-1 protein levels compared to cells

transfected with an empty vector (Figure 6D). These data suggest a possible role for miR-184 in the

pathophysiology of AMD.

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DISCUSSION

In this study we examined the role of miR-184 in regulating human RPE homeostasis. Our data

showed that miR-184 regulates EZR, a cytoplasmic peripheral membrane protein important for epithelial

cell morphology and function. Moreover, our data demonstrated that inhibition of miR-184 results in

down-regulation of LAMP-1 gene and protein expression and reduced phagocytic activity in human RPE.

Importantly, we investigated the role of miR-184 in RPE health and disease by analyzing RPE derived

from eyes of normal and AMD organ donors. MIR-184 was down-regulated in AMD RPE compared to

normal RPE and this was associated with LAMP-1 down-regulation.

The phagocytosis of the photoreceptors outer segment (POS) by RPE is required for eliminating

photoreceptor cell waste and retaining recyclable cellular material, and therefore is crucial for

maintenance of photoreceptors [49]. An age-related decline in phagocytosis is believed to contribute to

AMD [50, 51]. Here, by measuring the uptake of POS, we showed that inhibition of miR-184 reduced the

efficiency of POS uptake in ARPE19. EZR protein functions by directly binding to proteins including

LAMP-1 [31], and the molecular interaction of EZR with LAMP-1 is required for the formation of

phagocytic vacuoles [34]. Therefore, we speculate that inhibition of miR-184, translating into increased

EZR and decreased LAMP-1 expression, could lower their ability to interact. This could consequently

result in reduced phagocytic activity by inhibiting the formation of phagocytic vacuoles. In addition,

lower LAMP-1 protein levels could decrease phagolysosomal digestion and thus increase the amount of

undigested material remaining in the cells. The precise mechanism by which miR-184 regulates the

phagocytosis pathway and LAMP-1 expression is the subject of future investigations.

It is noteworthy that the role miRNAs play in post-translational regulation of different human

genes have only been recently investigated, yet there is an increasing interest in using miRNAs as

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diagnostic disease markers and for therapeutic approaches [7, 45]. With regards to AMD, miRNAs have

recently become a focus of therapeutic investigations and to date, no miRNAs have been directly

associated with the pathophysiology of AMD [48]. miR-184 has been linked to several diseases such as

EDICT syndrome, a hereditary eye disease, and familial keratoconus with cataract caused by a single

base mutation in the MIR-184 seed sequence [52, 53]. In addition to genetic mutations, defects in

epigenetic regulation pathways such as DNA methylation can also result in disease phenotypes. In the

case of Rett syndrome, a mutation in the methyl CpG-binding protein 2 (MeCP2) disrupts the silencing of

MIR-184 expression leading to up-regulation of miR-184, resulting in defective synaptic plasticity in

neurons [3]. miR-184 has also been shown to play an important role in adult neural stem/progenitor cell

differentiation. It is reported that methyl-CpG binding protein 1 (MBD1) directly represses MIR-184 in

adult neural stem/progenitor cells (aNSCs). In addition, MBD1 deficiency causes miR-184

overexpression and prevents aNSC differentiation. The inhibition of miR-184 can rescue phenotypes

associated with MBD1 deficiency [4]. The loss of expression may also lead to various deleterious

phenotypes just as overexpression of a microRNA can result in disease [45].

miR-184 is the most abundant miRNA in the central corneal epithelial basal, suprabasal cells

and in the lens epithelium [2, 54]. It is reported that miR-184 antagonizes the binding of miR-205 to

mRNA of the inositol polyphosphate phosphatase-like 1 gene (INPPL1 also known as SHIP2) [55], and

therefore prevents knock-down by miR-205 and rescues INPPL-1 production. Through this action, miR-

184 sustains the levels of phosphorylated AKT and phosphorylated BCL-2-associated death promoter

(BAD), which regulate apoptosis [53, 55]. This observation suggests that miR-184 may directly regulate

other regulatory factors, including other microRNAs, to maintain the expression of RPE genes critical for

cell survival and function. These studies and our novel data presented here support a model for miR-

184-dependent coordination of pro-survival signaling and critical membrane trafficking events for the

maintenance of healthy RPE.

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Increasing evidence suggests miRNAs as modulators of pathways related to inflammation,

cancer, cellular senescence and age-related diseases [56]. Our observation of MIR-184 down-regulation

in RPE from AMD donors further supports a critical role for miRNAs in pathophysiological processes. We

are currently investigating the direct association of loss of miR-184 expression with the pathophysiology

of AMD. In all, our observations suggest that miR-184 may serve as a potential biomarker and a

therapeutic target for treatment of this devastating neurodegenerative disease.

MATERIALS AND METHODS

Proteomics: miR-184 expressed in RPE cells was inhibited with 10 pmoles of hsa-miR-184 mirVanaTM

miRNA Inhibitor (Invitrogen, Carlsbad, CA) per 500,000 cells. The control cells were transfected with

High GC Duplex scrambled siRNA (Invitrogen). The cells were incubated for 72 hrs at 37°C at 5% CO2.

Proteins were extracted with RIPA lysis buffer (Cell Signaling, Danvers, MA), and precipitated with cold

acetone overnight at -20°C. The precipitate was digested with trypsin at 37°C for 1 hour after reduction

by tris 2-carboxyethyl phosphine (Sigma-Aldrich, St. Louis, MO) at 60°C and alkylation by methyl

methanethiosulfonate (Thermo Scientific, Waltham, MA) at room temperature. The controls and the

treated were compared in triplicates using a quadruplet iTRAQ labeling kit which allows relative

quantification of all identified proteins based on an introduced isotopic label. The samples seprated for

60 minutes by offline 2D chromatography on a HP 1100 HPLC system (Agilent Technologies, Waldbronn,

Germany) with XTerra MS C18 3.5µm column, 2.1x100mm (Waters, Milford, MA). The size and relative

quantity of the protein fragments were detected by subsequent QTOF mass spectrometric analysis (AB

Sciex, Foster City, CA).

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RPE cultures: Human primary cultures of RPE cells were established in our lab from eyes of organ

donors following a protocol previously described [57]. The primary RPE were cultured in EpiCM

epithelial cell medium (ScienCell) as described previously [58]. The Adult RPE (ARPE19) cell line was

generously provided by Dr. T. Michael Redmond’s lab (National Eye Institute, National Institutes of

Health, Bethesda, MD). The ARPE19 cells were cultured in high-glucose Dulbecco’s Modified Eagle

Medium (DMEM) with sodium pyruvate (Invitrogen) supplemented with 10% fetal bovine serum (FBS)

and 1% antibiotic/antimycotic and incubated at 37°C and 5% CO2. For phagocytosis and co-localization

experiments, ARPE19 cells were grown for 2 weeks post-confluency on 8 well chamber slides (Millicell EZ

slide, Milllepore, Billerica, MA) prior to experimentation.

Isolation of Photoreceptor Outer Segments: POS were generously provided by Dr. T. Michael Redmond’s

lab (National Eye Institute, National Institutes of Health, Bethesda, MD). POS were isolated and purified

as described previously [59] using a discontinuous density gradient method. Approximately 200 bovine

retinas were vigorously shaken in 180mL sucrose buffer (45% sucrose in buffer A: 100 mM potassium

phosphate, pH 7.0, containing 1mM MgCl2, 0.5mM DTT, and 0.1mM EDTA) followed by centrifugation at

3,000 g for 5 minutes and 4°C. The supernatant was then filtered through gauze, diluted 1:1 with buffer

A, and centrifuged at 4,400 g for 7 minutes to isolate the crude POS fraction. The supernatant was then

discarded and the pellet resuspended in 1mL of buffer A (density = 1.105). POS were then purified with

discontinuous density gradient centrifugation. Gradients were prepared in 50mL centrifuge tubes with

18mL sucrose in buffer A (density = 1.135), then 17 mL sucrose in buffer A (density = 1.115), and crude

POS layered on top. Tubes were then centrifuged for 1 hour at 27,000 g and 4°C in a Beckman SW28

rotor (Brea, CA) without the brake. POS were collected at the 1.115-1.135 interface, diluted 1:1 with

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buffer A, and harvested by centrifugation at 39,000g and 4°C. Pellets were stored at -80°C. Before use,

POS were resuspended in 100 mM bicarbonate buffer (pH 8.0) with 10% w/v sucrose, counted on a

hematocytometer and used at 2 x l06/ml.

Semi-quantitative and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR): For the analysis of

expression of human LAMP-1, EZR, AXL and TYRO-3 genes by qRT-PCR, total RNA was extracted from

RPE with the RNeasy kit (Qiagen, Germantown, MD), treated with RNase-free DNase I (Qiagen), and

reverse-transcribed with oligo-dT using the SuperScript III cDNA synthesis kit (Invitrogen) on a Veriti 96

well Thermal Cycler PCR Machine (Applied Biosystems, Foster City, CA). Semi-quantitative PCR was

performed using Taq Polymerase (New England Biolabs) for 25 cycles and the PCR products were

analyzed on a 2% agarose gel. Quantitative PCR was performed with the QuantiTect SYBR Green PCR Kit

(Qiagen). Specific primers for each gene were designed with the PrimerQuest software (Integrated DNA

Technologies, Skokie IL) and the cDNA sequences of each gene (GenBank) to produce 100-250 bp PCR

amplicons that span one or more exon/intron boundaries. Human GAPDH gene expression was

analyzed in parallel, for normalization. For the analysis of expression of MIR-184, microRNA samples

isolated from cultured RPE or HeLa cells using the mirVana miRNA isolation kit (Invitrogen), were reverse

transcribed and amplified with the miRCURYTM Universal RT microRNA cDNA PCR kit (Exiqon, Woburn,

MA). Quantitative PCR was performed with the miRCURYTM Universal RT microRNA PCR SYBR Green

Master Mix (Exiqon, Woburn, MA) and MIR-184 amplification primers (Qiagen). The expression of

human 5S rRNA gene was analyzed in parallel for normalization. The normalized relative expression

levels for each gene were calculated with the ΔΔCt method [60].

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Transfection assays: Nucleofections of plasmid DNAs, siRNAs or the miRNA inhibitors in HeLa or RPE

cells were performed with the Amaxa Biosystems Nucleofector II (Lonza, Allendale, NJ). 1 microgram of

plasmid DNA, 90 pmoles of control siRNA or 10 pmoles of the miRNA inhibitors (hsa-miR-184 or hsa-

miR-204 mirVanaTM miRNA inhibitors) were used to transfect 5x105 cells using the high efficiency I-013

protocol and the Basic Nucleofector Kit for Primary Mammalian Epithelial Cells Solution Mix (Cat. No

VPI-1005) from Lonza. To study the effect of mir184 inhibition on phagocytic activity and co-localization

of ezrin and LAMP-1, scrambled siRNA or the mir184 inhibitor were used to transfect cells seeded on 8

well chamber slides and grown to post-confluency for 2 weeks, using Lipofectamine 2000 (Invitrogen).

The concentration of lipofectamine used for transfection (0.35 μl per well) was optimized to permit the

highest transfection efficiency with the least cell death (<10%).

Luciferase Assays: HeLa cells were co-transfected by nucleofection with: a) a plasmid containing the

human MIR-184 gene in the pEZX-M04 vector, and b) a Luciferase-EZR-3’UTR construct in pEZX-M01

vector (both from GeneCopoeia, Rockville, MD). The bioluminescence from the expression of firefly

luciferase normalized to renilla luciferase for cell quantity variation, was quantified by MicroLumatPlus

LB 96V (Berthold Technologies, Oak Ridge, TN) in the absence and presence of ectopically expressed

MIR-184 gene.

Antibodies: Rabbit anti-beta actin (Cell Signaling, Danvers, MA), mouse anti-LAMP-1 (BD Biosciences,

San Jose, CA and EnCor Biotechnology, Gainesville, FL), rabbit anti-EZR (Cell Signaling, Danvers, MA),

Rabbit anti-TYRO-3 (Cell Signaling, Danvers, MA), goat anti-Rhodopsin (Santa Cruz Biotechnology Dallas,

TX) and rabbit anti-ZO-1 (Life Technologies, Grand Island, NY) (primary antibodies); Goat anti-rabbit IgG-

HRP linked, goat anti-mouse IgG-HRP linked (Cell Signaling, Danvers, MA), donkey anti-mouse Alexa 594,

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donkey anti-rabbit Alexa 594, donkey anti-rabbit Alexa 488 and donkey anti-goat Alexa 488 (Life

Technologies) (secondary antibodies).

Immunoblot Analysis: Protein samples were extracted in radioimmunoprecipitation assay (RIPA) buffer

(1% NP40, 0.5% sodium deoxycolate and 1% SDS in 1x Phosphate Buffered Saline), containing freshly

added Protease and Phosphatase Inhibitor Cocktail Tablets (Roche Applied Science), 1x Protease

Inhibitor Cocktail Set I (EMD Millipore), 1mM sodium vanadate, 50mM sodium fluoride, 1mM Phenyl

Methane Sulphonyl Fluoride (PMSF) (Sigma Aldrich). Protein concentrations were measured by

Bradford assay (Bio-Rad) on an Ultramak Microplate Imaging system (Bio-Rad, Hercules, CA).

Denatured protein samples were separated using the NuPAGE electrophoresis system (Novex 4-12% Bis-

Tris gels from Invitrogen) and transferred to an AmershamTM HybridTM-ECL nitrocellulose membrane (GE

Healthcare, Little Chalfont, UK) using the XCell western blot system (Life Technologies). Primary

antibodies were diluted 1/1000 in 5% BSA-1x TBST and incubated for 16-20 hrs at 4oC, and secondary

antibodies were diluted 1/3000 in 5% non-fat milk – 1x TBST and incubated for 90 min at room

temperature, based on the manufacturer’s instructions. Immunoreactive protein bands were visualized

by the SuperSignal® West Dura Chemiluminescent Substrate (Pierce, Rockford, IL) followed by imaging

with the myECL imager (Thermo Scientific Inc., Waltham, MA).

Immunofluorescence: ARPE19 cells were fixed with 4% paraformaldehyde for 10 minutes at room

temperature followed by ice-cold methanol for 1 minute. Cells were then permeabilized with 0.5%

Triton X for 5 minutes and blocked with BlockAid blocking solution (Life Technologies) for 1 hour at room

temperature before incubation with the primary antibodies in blocking solution overnight at 4°C. For

co-localization, primary antibodies used were rabbit IgG anti-Ezrin (1:100) and mouse IgG anti-LAMP-1

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(1:100). AlexaFluor488-conjugated donkey anti-rabbit (1:500) and AlexaFluor594-conjugated donkey

anti-mouse IgG (1:500) were used as secondary antibodies. For phagocytosis assay, primary antibodies

used were goat anti-rhodopsin (1:50) and rabbit anti-ZO-1 (1:100). AlexaFluor594-conjugated donkey

anti-rabbit IgG (1:500) and AlexaFluor488-conjugated donkey anti-goat IgG (1:500) were used as

secondary antibodies. Nuclei were counterstained with NucBlueTM Fixed Cell Stain (Life Technologies).

After staining, chamber slides were mounted with ProLong Gold Antifade mounting solution (Life

Technologies) and imaged using an Olympus BX61 Laser Scanning Microscope (Melville, New York).

Single x-y scanned images were used to count POS numbers and assess EZR - LAMP-1 co-localization.

Microscopic panels were composed using Adobe Photoshop CS4 (Adobe, San Jose, CA).

Phagocytosis Assays: The ARPE19 monolayers were transfected with either the hsa-miR-184 mirVanaTM

miRNA inhibitor or control scrambled siRNA for 48 hrs after being cultured for two weeks to post-

confluency in 8-well chamber slides. POS were added at 44 hrs at a concentration of 10 POS particles

per cell. After 4 hrs incubation with POS cells were washed with PBS, fixed, and imaged as described

above. Three independent experiments were carried out and POS particles were counted using ImageJ

software (http://rsb.info.nih.gov/ij/index.html) in quadruplicates for each well (n=3).

Immunoprecipitation: ARPE19 were lysed in RIPA buffer as described above and immediately processed

for immunoprecipitation with the anti-EZR antibody. The protein samples were pre-cleared with

protein-A agarose beads following the manufacturer’s instructions (Cell Signaling). Protein

concentration was measured by the Bradford assay (Bio-Rad, Berkley, CA) and was adjusted at 1mg/ml.

Homogenates containing 300 μg of total protein were incubated with the antibody to EZR diluted 1:1000

for 16 hrs at 4oC, and then protein-A agarose beads were added for 2 hrs. The immunoprecipitates (IP)

were rinsed in lysis buffer three times and then subjected to immunoblot analysis with anti-EZR and

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anti-LAMP-1 antibodies. The ‘input’ protein lysates were also analyzed by immunoblot with anti-beta

actin for cell count normalization.

Protein Quantitation Densitometry: In order to quantitate the protein results obtained by Western blot

analysis, we used ImageJ densitometry gel analysis software and protocol

(http://rsb.info.nih.gov/ij/index.html) with three replicate gel runs. The area calculated for each protein

run was normalized with the area for beta actin run in the same gel.

Statistical Analysis: To obtain statistically significant data from Proteomics, Phagocytosis assays,

Luciferase Assays and qRT-PCR, the experiments were performed in replicas of n=3-6. The results were

presented as mean ± Standard Deviation. The statistical analysis was performed with a two-tailed

Student’s t-test for two sample equal variance. P-values inferior to 0.05 indicated statistical

significance.

ACKNOWLEDGEMENTS – AUTHOR CONTRIBUTIONS

The Georgetown-Lombardi Comprehensive Cancer Center Shared Resource facilities were employed for

the proteomics analysis and qRT-PCR instrument use. We thank Dr. T. Michael Redmond, Chief

Laboratory of Retinal Cell and Molecular Biology (LRCMB), NEI/ NIH, Drs. Eugenia Poliakov, and William

Samuel, Staff Scientists, LRCMB, NEI/NIH for the generous gift of POS and low passages of ARPE19 cells.

Najiba Murad performed experiments, analyzed data and edited the manuscript.

MK, Nishantha Gunawardena, MS.G, YH, KJJ and ACT performed experiments and analyzed data;

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MK and ACT edited the manuscript.

Nady Golestaneh, designed the experiments, analyzed data and wrote the manuscript.

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Table 1. The clinically diagnosed AMD and control donors from which primary RPE cultures were derived.

Donor ID Clinical Diagnosis Gender Age (yrs)

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Ctrl 6 Normal Male 72





AMD 9 AMD Female 68

AMD 14 AMD Male 82

AMD 17 AMD Male 81

AMD 19 AMD Female 80

AMD 32 AMD Female 75

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Figure 1. miR-184 is evolutionary conserved and expressed in human RPE. A. Alignment of the miR-

184 nucleotide sequences of various species from insects to humans revealed that miR-184 is highly

conserved especially in the seed sequence (underlined). B. Proteomics data of RPE transfected with the

mir-184 inhibitor or with scrambled siRNA (negative control). The 5 most dysregulated proteins are

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shown, including EZR. C. qRT-PCR showing that human MIR-184 is expressed in human native RPE, adult

ARPE19 cell line, but not in the HeLa cell line.

Figure 2. miR-184 binds to the EZR mRNA 3’UTR and regulates the expression of EZR. A. Luciferase

assay performed on HeLa cells transfected with Luciferase-EZR 3’UTR construct with and without

ectopically expressed MIR-184, showing significantly reduced luciferase activity in the co-transfected

HeLa cells (p-value < 0.01, n=6). B. Luciferase assay performed on ARPE19 cells transfected with

Luciferase-EZR 3’UTR construct with and without treatment with miR-184 inhibitor (hairpin) showing

significantly increased luciferase activity in the co-transfected ARPE19 cells (p-value < 0.01, n=6). C.

Human EZR mRNA 3’UTR containing a putative binding site (underlined) for the miR-184 seed sequence.

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Figure 3. miR-184 affects phagocytosis in human RPE. A. qRT-PCR analysis 48 hrs after transfection of

ARPE19 with miR-184 or scrambled siRNA showing knockdown of MIR-184 (p-value<0.05, n=3). B.

Graphical representation of the phagocytosis assay performed in human adult RPE (ARPE19) transfected

either with miR-184 inhibitor or with scrambled siRNA and incubated with photoreceptor outer

segments (POS) for 4hrs. ARPE19 from three independent transfection experiments were

immunostained with anti-rhodopsin and anti-ZO-1 and analyzed by confocal microscopy. Single x-y

scanned images (n=11) from each transfection experiment were used to count the rhodopsin-positive

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POS. Phagocytosis efficiency is presented as the average numbers of uptaken POS per 100 cells and

standard deviations are shown, demonstrating significantly reduced phagocytosis efficiency in the

presence of the miR-184 inhibitor, 48 hrs after transfection (p-value < 0.05, n=11). Asterisks in A and B

indicate statistical significance as determined by the t-test (p-value < 0.05). C-D. Immunostaining with

anti-ZO1 (red) and anti-Rhodopsin (green) reveals the uptake of POS (white arrows) by cells in both the

control (C) and transfection with miR-184 inhibitor (D). Nuclei are counterstained with DAPI (blue).

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Figure 4. miR-184 modulates the expression of EZR and LAMP-1. A. qRT-PCR analysis 72 hrs after

transfection of ARPE19 with the miR-184 or miR-204 inhibitors showing complete silencing of MIR-184

(p-value=0.02, n=3) or MIR-204 genes, respectively. B. Semi-quantitative RT-PCR analysis performed on

mRNA from ARPE19 cells 48hrs after their transfection with scrambled siRNA or with the miR-184

inhibitor showing up-regulation of EZR expression and down-regulation of LAMP-1. TYRO3 and AXL

expression does not change in this experiment. C-D. qRT-PCR analysis performed on mRNA from ARPE19

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cells 48hrs (C) and 72 hrs (D) after their transfection with scrambled siRNA, or with miR-184 inhibitor, or

miR-204 inhibitor, showing significant decrease of LAMP-1 expression specifically by the miR-184

inhibitor at 48hrs (p-values < 0.01, n=3) (C) and up-regulation of EZR by both miR-184 and miR-204

inhibitors at 72hrs (p-values < 0.01, n=3) (D). AXL and TYRO3 expression levels were not significantly

affected (D). E. Western blot analysis on protein samples isolated from human ARPE19 cells transfected

with scrambled siRNA or with the miR-184 inhibitor, showing reduced LAMP-1 and increased Ezrin

protein expression. TYRO3 levels were not affected. F. The quantitation by ImageJ densitometry of

multiple Western blot analyses for LAMP-1, EZR and beta actin protein levels is also shown (LAMP-1: p-

value=0.01, n=3, EZR: p-value<0.01, n=3). Asterisks indicate statistically significant differences in protein

levels determined by t-test (p-value<0.05).

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Figure 5. miR-184 promotes LAMP-1 expression and interaction with EZR. A-D: Representative

confocal images (single x-y scans) of ARPE19 transfected with scrambled siRNA (A and C) or miR-184

inhibitor (B and D) and co-stained with anti-LAMP-1 (red), anti-EZR (green) and DAPI (blue) 48 hrs after

transfection. Images in (A) and (B) show LAMP-1 and DAPI staining; (C) and (D) show EZR (green)

merged to the images (A) and (B), respectively. Transfection of the miR-184 inhibitor significantly

decreased LAMP-1 expression, as revealed by the reduced red fluorescence in (B) compared to (A).

Consequently, the LAMP-1 – EZR co-localization was decreased by the miR-184 inhibitor, as shown by

the reduced number of co-localization sites in (D) compared to (C). (E) and (F) are histograms of color

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intensity and distribution generated in PhotoShop for the images in (C) and (D) respectively, showing the

reduction in the amount of co-localization (yellow and grey) in (F) compared to (E). Three independent

transfection experiments were performed and 10 randomly selected images were observed for co-

localization. The results are represented by the images shown here. G. Immunoprecipitation analysis of

protein samples from ARPE19 transfected with the miR-184 inhibitor or scrambled siRNA, followed by

precipitation with anti-EZR. EZR-bound LAMP-1 (IP) as well as total LAMP-1 protein (Input) were

significantly decreased in the presence of the miR-184 inhibitor. Ezrin level in the IP precipitate was

increased. Beta actin levels are shown for normalization. H. Densitometry analysis of the

immunoprecipitation in (G). Asterisks indicate statistically significant differences, as determined by the

t-test (p-value<0.05).

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Figure 6. MIR-184 is down-regulated in the RPE of donors with AMD. A. qRT-PCR analysis of MIR-184,

MIR-204 and LAMP-1 in mRNA from five normal (control) primary RPE cultures and five AMD RPE

cultures, showing significant down-regulation of MIR-184 in the AMD RPE while the MIR-204 levels do

not show a significant difference between AMD and control RPE. LAMP-1 mRNA levels correlate with

MIR-184 expression, being the lowest in the AMD RPE cultures. B-C. Expression of MIR-184 (B) and

LAMP-1 (C) was restored in the AMD 9 and AMD 14 RPE, following 72hrs transfection with the MIR-184

overexpressing plasmid, but not with the empty vector, as shown by qRT-PCR analysis. The reactions in

A, B and C were performed in triplicates. Asterisks in (A) indicate significantly lower expression levels in

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the AMD samples compared to control samples, as determined by the t-test (p-values < 0.05, n=3).

Asterisks in (B-C) indicate statistically significant increase in expression levels of MIR-184 (B) and LAMP-1

(C) after transfection of AMD 9 and AMD 14 RPE with the MIR-184 overexpressing plasmid (p-values <

0.05, n=3). D. Western blot analysis of the AMD 9 and AMD 14 RPE transfected with either the MIR-184

overexpressing plasmid or with an empty vector, showing restoration of normal LAMP-1 protein levels

only in the presence of the MIR-184 overexpressing plasmid. Normal RPE cultures (Ctrl 6 and Ctrl 25),

transfected with an empty vector are shown for comparison. Beta actin expression is used for

normalization.

miR-184 regulates ezrin, LAMP-1 expression, affects phagocytosis in human retinal pigment epithelium...

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