In vertebrates, the RPE originates from the dorsal portion of the optic cup, while the retina and the optic stalk develop from the distal/ventral portion (Amram et al., 2017). Once specified, the RPE cells begin to differentiate, changing in size and shape. With the folding of the optic cup, RPE progenitor cells constitute a ciliated and pseudostratified epithelium, which is committed into a cuboidal epithelium following the formation of interphotoreceptor matrix. As eye morphogenesis proceeds, the presumptive RPE tissue assumes an apical to basolateral polarity and forms both microvilli and tight junctions. On further differentiation, the RPE cells adopts a hexagonal shape, with elongated microvilli and a smooth basal surface which makes continuous contact with its basement membrane (Marmorstein et al., 1998). In adults, RPE tissue comprises a monolayer of polarized and pigmented epithelial cells that resides at the interface between the light-sensitive outer segments of the PR and vessels of choriocapillaris (Strauss, 1995, 2005). The RPE layer is a critical constituent tissue capable of absorbing the light energy focused by the lens on the retina, thus mitigating photo-oxidative stress (Bok, 1993). In addition, the RPE represents part of the tight retinal–blood barrier (Simo et al., 2010); supports the isomerization of all-trans-retinal to 11-cis-retinal, the visual chromophore required for photoreceptor excitability (Strauss, 2005); protects from oxidative stress (Strauss, 2005); secretes growth factors that help to maintain the structural integrity of photoreceptors and choriocapillaris endothelium (Strauss, 2005; Mazzoni et al., 2014); establishes ocular immune privilege by expressing immunosuppressive factors (Ao et al., 2018); and finally is critical in the continuous renewal of the photoreceptors outer segments (POS), which are regularly shed by phagocytosis, to rebuild light-sensitive outer segments from the base of the photoreceptors (Mazzoni et al., 2014). To realize these complex functions, intricate molecular networks, activated by extracellular and intracellular signals, are simultaneously employed to maintain RPE homeostasis and function. Due to the significant activity of microRNAs (miRNAs) in modulating essential biological processes by targeting networks of functionally correlated genes, it is unsurprising that miRNAs have emerged as indispensable components of these molecular networks in the RPE (Soundara Pandi et al., 2013; Greene et al., 2014). Importantly, alterations to gene regulatory networks controlling any of the above activities of the RPE can lead to degeneration of the retina and loss of visual function. This in turn gives rise to diseases including retinitis pigmentosa, age-related macular degeneration (AMD), and other blindness conditions in humans (Strauss, 2005; Amram et al., 2017).

The gene regulatory networks involved in establishing the RPE development and differentiation have been extensively studied in a variety of model organisms reviewed in Amram et al. (2017), but the critical roles of miRNAs in these processes have only recently been explored. Recent studies have reported miRNA expression profiles during RPE development and differentiation in a variety of species, using multiple approaches including next generation sequencing (NGS) technology and in situ hybridization (ISH) analysis (Deo et al., 2006; Xu et al., 2007; Damiani et al., 2008; Decembrini et al., 2009; Arora et al., 2010; Georgi and Reh, 2010; Hackler et al., 2010; Karali et al., 2010; La Torre et al., 2013; Wohl and Reh, 2016). Importantly, NGS analyses showed dynamic miRNA expression profiles during RPE differentiation, indicating a strict correlation between miRNA expression levels and the stages of iPSC-RPE differentiation (Wang et al., 2014). To characterize the roles of miRNAs in the RPE development, depletion of the enzymes essential for their maturation and processing were examined in several animal models. Conditional knockout (cKO) mice, for Dicer1, Drosha, or Dgcr8 genes in the RPE, demonstrated that this class of proteins is relevant for RPE differentiation and maintenance. Dicer1 cKO in the ocular pigmented cell lineage around postnatal (PN) day 9.5, generated by crossing Dgcr8^LoxP/LoxP animals with Dct-Cre, Tyrp2-Cre, and α-Cre mice, provided the earliest evidence of the impact of simultaneous loss of miRNAs in the generation and survival of non-retinal cell types (Davis et al., 2011). Consistent with these findings, Dicer1 cKO at the optic vesicle stage (E9.5), using Dct-Cre mice, revealed alterations to RPE differentiation. At PN11 days, Dicer1^loxP/loxP/Dct-Cre mice showed RPE cells that were smaller than normal, depigmented, and failed to express enzymes required for the visual cycle (Ohana et al., 2015; Amram et al., 2017). However, RPE cell fate and hexagonal cell morphology were preserved, as was the conserved expression of transcription factors (i.e., OTX2, MITF, and SOX9), which participate in RPE specification and maintenance (Amram et al., 2017). Similar phenotypes were also observed in both RPE-specific Drosha and Dgcr8 cKO mouse models (Ohana et al., 2015; Amram et al., 2017). Importantly, the maintenance of RPE cell fate was further corroborated by the transcription profile of Dicer1-deficient RPE cells. These data gave rise to the hypothesis that RPE fate might be acquired and maintained through miRNA-independent mechanisms. Together, these findings supported the notion that miRNAs were necessary for the execution of proper differentiation programs during RPE development (Ohana et al., 2015; Amram et al., 2017). In contrast to the findings on the role of Dicer1 in miRNA biogenesis during RPE differentiation, a non-canonical Dicer1 activity was demonstrated to participate in the adult RPE from Dicer1^loxP/loxP/Best1-Cre mice, with Cre recombinase expression beginning at P10 and peaking at P28 (Iacovelli et al., 2011). In differentiated RPE cells, a primary role for Dicer1 was documented in the degradation of toxic transcripts of Alu transposable elements, rather than miRNA biogenesis (Iacovelli et al., 2011). Supporting this notion, single miRNA-binding protein knockout mice, for Ago1, Ago3, Ago4, or Tarbp2, did not reveal RPE morphological alterations (Sundermeier and Palczewski, 2016). Consistent with these findings, AAV-mediated delivery of a Best1-Cre recombinase expression cassette into mice carrying conditional Drosha, Dgcr8, or Ago2 alleles did not show any defects in RPE morphology (Sundermeier and Palczewski, 2016). However, lack of miRNA biogenesis in both differentiating and adult RPE commonly affected the homeostasis and survival of the adjacent photoreceptor cells. Nevertheless, to gain deeper phenotypic understanding, additional studies should analyze alternative miRNA biogenesis mechanisms and the compensatory molecular networks that counteract lack of enzymes essential for miRNA maturation and processing in the RPE (Yang and Lai, 2011).

The RPE exhibits common features of an epithelium tissue, preserving the structural and physiological integrities of adjacent tissues. The RPE plays crucial roles in retinal homeostasis, including the formation of the outer blood–retinal barrier (BRB), through junctional complexes, which prevents the entry of toxic molecules and plasma components into the retina (Rizzolo, 2007). Alterations to RPE physiology and homeostasis result in photoreceptor death and blindness. As part of the BRB, the RPE transports water, ions, and metabolic waste from the subretinal space to the choriocapillaris (Marmor, 1990; Hamann, 2002), where it takes up nutrients such as glucose or vitamin A, retinol, and fatty acids to the photoreceptors (Ban and Rizzolo, 2000; Marmorstein, 2001). The RPE secretes different growth factors according to variable physiological and pathological conditions. During eye development, growth factors released from RPE play key roles in choroidal neovascularization. To regulate transport across the RPE, numerous pumps, channels, and carriers are specifically distributed to either the apical or the basolateral membrane. Transporters and channels with roles in the selective transport to and from the choroid vasculature are located in the basal RPE. The apical side of the RPE projects long thin and sheet-like microvilli into the interphotoreceptor matrix. The long microvilli engulf the tips of the rod and cone photoreceptor outer segments; this ensures that the segments are orientated in the appropriate plane for optics and maximizes the cellular surface for efficient epithelial transport. The short microvilli mainly function in the turnover of POS through phagocytosis. Moreover, RPE cells are constantly exposed to oxidative stress due to exposure to light and the generation of reactive oxygen species (ROS) following phagocytosis of POS (Plafker et al., 2012). A robust endogenous antioxidant defense mechanism is therefore critical. This is characterized by the production of endogenous antioxidants such as glutathione, whose production decreases with age and/or incidence of degenerative diseases (Beatty et al., 2000). Oxidative stress alters RPE integrity and promotes the activation of the inflammation system, activating complementary cascades and upregulating the production of cytokines and chemokines. Notably, the mature RPE itself maintains tissue homeostasis through long-term cell survival, with little evidence of cell turnover and a stem cell compartment for de novo cell production. This static homeostasis suggests that as RPE cells age, substantial tissue changes occur that compromise RPE function and morphology, triggering causes of AMD (Fuhrmann et al., 2014). Based on its structural characteristics, the RPE plays crucial roles in retinal homeostasis, of which a primary role is the formation of the outer BRB that prevents the entrance of toxic molecules and plasma components into the retina (Rizzolo, 2007). Breakdown of the BRB can lead to visual loss in a number of ocular disorders, including diabetic retinopathy (DR).

Vision relies on the functional interaction between RPE and photoreceptors. RPE takes an active part in the visual cycle, as it expresses the enzymes required for reisomerization of the 11-cis chromophore from all-trans-retinal, which is essential for initiating the photoreceptor response to light. All-trans-retinal is reduced to all-trans-retinol within the photoreceptors and then transported to the RPE, where it is esterified by LRAT (Saari and Bredberg, 1989; Ruiz et al., 1999). The all-trans-retinyl ester product is then isomerized by RPE65 and hydrolyzed to release 11-cis-retinol (Redmond et al., 2005). Importantly, phagocytosis and autophagy are crucial cellular mechanisms required for maintaining RPE/PRs homeostasis and supporting the visual system. Light exposure to photoreceptors is accompanied by photo-oxidation of proteins and phospholipids of the outer segments (Beatty et al., 2000). To maintain their function, the POS are shed on a circadian basis (LaVail, 1976, 1980) and phagocyted by the RPE, where they fuse with lysosomes for the degradation and recycling of the ingested POS cargo (Anderson et al., 1978). Alterations to any of the above activities of the RPE is thought to be the primary cause in retinal pathologies, including AMD, because the RPE is vital for the health, survival, and function of the adjacent retinal PRs and choroid (Nandrot et al., 2004, 2006). Remarkably, oscillations in genes related to these cellular processes were observed to respond rapidly to changes in the light environment supporting combinatorial light-dependent and circadian-mediated regulation of visual POS turnover. Phagocytosis and degradation of POS occurs by a non-canonical form of autophagy termed LC3-associated phagocytosis (LAP), which exploits the effectors of autophagy in the process of phagocytosis (Kim et al., 2013; Ferguson and Green, 2014). Importantly, during LAP process, the phagocytic machinery takes advantage of autophagy components Atg5, Atg7, Atg3, Atg12, and Atg16l1 for the lipidation of LC3, resulting in LC3 recruitment to the phagosome and uses a BECN1-PIK3C3/VPS34 complex lacking Atg14. LAP is also independent of the autophagy preinitiation complex consisting of ULK1/Atg13/FIP200 (Kim et al., 2013). Engulfed POS enters the phagosome on a daily basis, the Atg12-Atg5-Atg16L1 complex is recruited, as is the lipidated form of LC3 (LC3-II). Only then does the lysosome fuse with the phagosome forming the phagolysosome leading to degradation of the POS cargo (Ferguson and Green, 2014).

Age-related macular degeneration is a multifactorial, degenerative disease and the most common cause of vision impairment and blindness in the elderly population, with approximately 30–50 million people affected worldwide. The susceptibility to develop “dry” and “wet” AMD forms is dependent on a combination of genetic components and environmental factors. AMD, as well as other forms of retinal degeneration, is often similar and associated with impaired function of the RPE. AMD is characterized by deposits of lipofuscin and extracellular protein aggregates called “drusen” determining progressive dysfunction of autophagy and both RPE and photoreceptor cell death. Interestingly, studies carried out on AMD highlighted that Dicer1 mRNA was reduced in the RPE, but not into neural retina, by 65 ± 3% compared to control eyes, while there was no differences in Drosha and Dgcr8 mRNAs in AMD eyes (Kaneko et al., 2011). To define the relevance of Dicer1 reduction in the RPE, Kaneko et al. (2011) crossed Dicer1f/f mice with BEST1 Cre mice, which express Cre recombinase under the control of the RPE-cell-specific BEST1 promoter. Results showed that these mice displayed RPE cell degeneration in comparison to controls. However, Dicer1 dysregulation in these mice was associated to Dicer1 capacity of silencing toxic Alu transcripts rather than miRNA biogenesis (Kaneko et al., 2011). To verify the hypothesis that miRNA dysregulation is implicated in the pathophysiology of AMD, global expression profiles of miRNAs have been investigated. Several studies examined + circulating miRNAs as well as tissue-specific miRNA expression patterns from age-matched control and affected individuals. The results show a number of discrepancies in the number and type of miRNA identified, probably due to the procedures applied to quantify miRNAs and selection of analyzed samples. In spite of these discrepancies, it is evident that most of miRNAs participated to oxidative stress, inflammation, lipid metabolism, and angiogenesis. Remarkably, miRNA expression profiles in both AMD mouse and rat models exhibited overlapping results, suggesting that the main role of miRNAs in RPE is to protect against oscillations in gene expression and counteract environmental stress, in an effort to preserve RPE cellular homeostasis (Berber et al., 2017). Table 4 lists the most commonly reported miRNAs identified in pathological RPE conditions. Interestingly, the most frequently altered expression identified was that of miR-146a, which occurred in both “dry” and “wet” AMD patients and was found in the RPE of different AMD animal models. Remarkably, in vivo and in vitro studies of specific miR-146a functions demonstrated key roles including repressing expression of IL-6 and VEGF-A genes and inactivating the NF-κB signaling pathway in the RPE. The latter, together with the gain-of function studies for miR-146a, suggests a crucial role for this miRNA in controlling genetic pathways essential to innate immune responses, inflammation, and the microglial activation state, which are major features in the pathogenesis of AMD (Hao et al., 2016). This further suggests that miR-146a may represent a useful disease biomarker and, additionally, a valuable therapeutic target for AMD treatment. Moreover, recent findings reported the role of the epithelial–mesenchymal transition (EMT) in the RPE as a pathological feature of the early stages of AMD (Shu et al., 2020). Remarkably, transforming growth factor-beta (TGFβ) has been identified as a key cytokine orchestrating EMT, and its alteration has been largely associated with onset and progression of several ophthalmological diseases, including AMD (Wang et al., 2019). Interestingly, dynamic changes in expression levels of several miRNAs were associated with TGF-β signaling during EMT in the RPE (Li D.D. et al., 2016). Among these, miR-29b was the most significantly downregulated. The function of the miR-29b was examined in ARPE-19. Additional consequences of miR-29b downregulation included the downregulation of E-cadherin and ZO-1, upregulation of α-smooth muscle actin (α-SMA), and increased cell migration. In vitro analyses showed that overexpression of miR-29b was sufficient to reverse TGF-β-mediated EMT through targeting Akt2. In agreement with this, silencing of the Akt2 abolished miR-29b-mediated repression of EMT process (Li M. et al., 2016). Similarly, a detailed study by Jun and Joo (2016) demonstrated that miR-124 was also involved in EMT in the ARPE-19. miR-124 overexpression induced occludin (OCLN) and ZO-1 and repressed α-SMA by directly targeting Ras homology growth-related (RHOG) (Jun and Joo, 2016). Besides miR-29b and miR-124, other miRNAs have been shown to negatively control the EMT. The levels of the ZEB1 protein, a key transcription factor in EMT, in ARPE-19 cells were decreased in the presence of miR-194 duplexes and elevated on miR-194 inhibition. In agreement with these observations, the levels of expression of ZEB1 target genes were reduced in response to miR-194 overexpression as a consequence of alterations in ZEB1 repression. The miR-194 targeting of ZEB1 has also been confirmed in vivo. Exogenous administration of miR-194 ameliorated the pathogenesis of proliferative vitreoretinopathy in a rat model (Cui et al., 2019). Accordingly, a recent study also showed that both miR-302d and miR-93 exert their functions in regulating TGFB signaling in the RPE (Fuchs et al., 2020). Altogether, these data highlight a potential use for miRNA: as a promising therapeutic resource for the treatment of ocular disorders. An additional key player involved in AMD and pathophysiology of RPE is miR-211. Indeed, two independent studies based on genome-wide searches for novel AMD risk factors identified miR-211 and its host gene, namely TRPM1, as a locus-containing risk factors for AMD (Persad et al., 2017; Orozco et al., 2020). Remarkably, the RPE phenotype in the miR-211^–/– mice was similar to that observed in animal models for AMD conditions. Moreover, miR-211 gene delivery via gene therapy was also explored to promote RPE and PR survival in AMD (Cunnusamy et al., 2012). These studies, together with in vitro studies demonstrating the function of miR-211 in the physiology of RPE, support that miR-211 is central to RPE homeostasis and, consequently, may serve as a precious molecular tool to counteract AMD disease. A number of additional examples of miRNAs involved in inflammation, oxidative stress, and angiogenesis have already been shown to be potential therapeutic targets for AMD. miR-23a is downregulated in RPE cells from AMD patients, and overexpression of this miRNA in ARPE-19 reduces cell apoptosis by regulating Fas (Lin et al., 2011). On the contrary, upregulation of miR-218 accelerates the apoptosis of ARPE-19 cells, negatively regulating Runx2. These finding reveal that miR-218/Runx2 axis could be a therapeutic target for retinal diseases (Yao et al., 2019) (Figure 4).

We are in the process of increasing our knowledge on the role of miRNAs as key regulators of RPE homeostasis, function, and survival. miRNAs play an important role in contributing to the precise control of RPE homeostasis in both physiological and pathological states. These insights are opening up avenues to a wide range of novel research areas, including gene-independent medicine, and offers an exclusive class of biomarkers for diagnostic analysis. Recent preclinical studies indicate that miRNAs-based gene therapy is nearing the transition to clinical trial stages. As a single miRNA has the potential to modulate and orchestrate entire molecular programs, future studies are required to shed new light on how they may be safely used as key factors stabilizing and/or resetting the RPE state in pathological conditions. However, recent findings suggest that they are highly promising molecular tools to treat and counteract retinal degeneration including the AMD onset and progression.

DI, GG, and IC have contributed to wrote the manuscript and prepared the figures. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.