MicroRNAs belong to the family of non-coding RNAs that participate in cell proliferation, cell death and development. The Müller glial cells are the inherent and specific neuroglia cells in the retinal organisation and play significant roles in retinal neuroprotection, organisational maintenance, inflammation and immunity, regeneration, and the occurrence and development of retinal diseases. However, only a few studies report the underlying mechanism of how miRNAs drive the function of Müller glial cells in the development of retinal diseases. This review aims to summarise the roles of miRNAs in retinal Müller glial cell function, including gliogenesis, inflammation and immunity, regeneration, the development of retinal diseases, and retinal development. This review may point out a novel miRNA-based insight into retinal repair and regeneration. MiRNAs in Müller glial cells may be considered a diagnostic and therapeutic target in the process of retinal repair and regeneration.
Data availability statement
The data sets used and analysed during the current study are included within the article and its additional files. All material used in this review are from publicly available articles.
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MicroRNAs (miRNAs) belong to a non-coding family of RNAs consisting of short sequences of approximately 22 nucleotides. miRNAs were initially reported to regulate the development of organisms,1 as indicated by the discovery of lin-4 and let-7. Functional mutations in these miRNAs directly affect the developmental timeline in Caenorhabditis elegans.2 3 Studies have reported the effects of miRNAs on the proliferation, death and development of Drosophila and Bantam cells.1 Until recently, miRNAs were recognised as regulatory molecules that are processed by the microprocessor complex into a precursor miRNA, which is then cleaved by the endoribonuclease Dicer1 into the mature and functional form. The miRNAs directly regulate gene expression by combining with mRNA complementary to the 3'-untranslated regions of genes and silencing the expression of target genes.1 The production of mature miRNAs in organisms must rely on Dicer, an RNase III enzyme required to produce mature miRNAs.4 In addition, functional miRNAs must combine with argonaute proteins to form a silencing complex to carry out their biological functions and then combine with the transcription sequence of a target gene to silence gene expression.5 In recent years, increasing evidence has suggested the involvement of miRNAs in Müller glial cells, which participate in various biological functions of the retinas.6 7 Based on the function of miRNAs in retinal maintenance and regeneration, it is important to consider miRNAs in Müller glial cells as diagnostic and therapeutic targets in the process of retinal repair and regeneration.
Multiple functions of retinal Müller cells
Müller cells are inherent glial cells in the retina derived from the same retinal progenitor cells (RPCs) as retinal neurones. The body of Müller cells is in the inner nuclear layer (INL) of the retina. The end processes of the cell protrusions extend into the outer and inner limiting membranes, penetrating the retina as a whole (figure 1). Under normal circumstances, similar to other glial cells in the central nervous system, Müller cells play a significant role in supporting and nourishing retinal neurons.8 9 After retinal injury or under retinal disorder circumstances, Müller cells can express glutamate transporter and glutamine synthetase to counteract glutamate-induced excitatory toxicity8 9 or express glutathione to relieve oxidative stress of the retina.8 9 In addition, Müller cells express neurotrophic receptors and release neurotrophic factors such as brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, ciliary neurotrophic factor, leukaemia inhibitory factor, nerve growth factor, and basic fibroblast growth factor to nourish and protect damaged retinal neurons.8 9 Apart from the protective effects of Müller cells, they may also serve as an important source of cytokines and inflammatory factors in response to retinal injury,10 further exacerbating the microenvironment of retinopathy and promoting neuronal death in the retina. Müller cells express major compatibility complex II antigens during inflammation and are involved in retinal immune responses.8 9 Moreover, during retinal inflammation, the morphologies of Müller cells and processes are significantly altered, accompanied by reactive gliosis, such as the upregulation of GFAP proteins.8 9 11 In postretinal injuries, Müller cells can dedifferentiate into RPCs and reprogram into neurones, which share the same progenitor cells as the Müller cells.8 The regeneration of Müller cells has been associated with Notch-3 and N-cadherin.9 Therefore, Müller cells play an important role in retinal function and maintaining homeostasis. The dysfunction of Müller cells is associated with many retinal diseases, such as macular telangiectasia and diabetic retinopathy, among others.12 13 Therefore, Müller cells are involved in neuroprotection, inflammation, immunity, retinal regeneration, development, and the progression of retinal disorders.
The miRNA expression profiles in Müller cells
To better understand the role of miRNAs in Müller cell function, it is necessary to identify miRNA expression profiles. The functions of Müller cells are modulated by miRNAs. Recently, expression profiles of miRNAs in Müller cells have been reported in several studies. More than 600 miRNAs have been identified in adult mice retinas.14 Of these, seven miRNAs (−204, −125b-5p, −9 to –99 a, −135a, −23a, and -100) were highly expressed in Müller cells and weakly expressed in neurones, 15 miRNAs were highly expressed in Müller cells and neurones, and many miRNAs were weakly expressed in Müller cells but highly expressed in neurones.14 Among the seven highly expressed proteins in Müller cells, most were upregulated during Müller cell maturation and development. However, after in vitro culture, 80% were downregulated, although the underlying mechanism for this phenomenon remains unclear.14
To search for candidate miRNAs involved in Müller cells in the developing retina, Quintero et al compared miRNA expression profiles among late RPCs, CD73 positive (+) rods, and postnatal Müller cells.15 The miRNA expression profiles were very similar between RPC and CD73+ rod photoreceptors but different between CD73+ rod and Müller cells. Specific miRNAs in Müller cells are miR-143, 145, 214, 199a-5p, and 199b.15 In addition, the authors found decreased expression levels of miR-143 and 145 but increased levels of miR-29a in Müller cells derived from RPCs. In addition, miR-124a, miR-9, and miR-181c were upregulated during the appearance of Müller cells derived from RPCs differentiating into rods.15 Similar studies have been reported in zebrafish, in which the knockdown of Dicer in the retina significantly reduced the number of proliferating Müller cells derived from RPCs during regeneration and was accompanied by the downregulation of several miRNAs, including miR-142b, miR-146a, miR-7a, miR-27c, and miR-31. The inhibition of the expression of these miRNAs also reduces cell proliferation in the INL (where the somas of Müller cells are located).16
Therefore, the identified miRNAs may be potential targets for gliogenesis and reprogramming Müller glial cells in the mammalian retina.
MiRNAs are involved in Müller glial gliogenesis
Multiple miRNAs are associated with gliogenesis in Müller cells. The role of miR-7a in mouse retinal development was reported by Baba et al. The results revealed that the expression of cyclin D3, a marker of Müller cells, was downregulated after the overexpression of miR-7a in the P0 mouse retina and was upregulated after miR-7a downregulation. Further experiments suggested that miR-7a targets Notch3, thereby attenuating Notch signalling to promote the differentiation of Müller cells from RPCs.17 Additional studies have shown that the expression of let-7 is persistently upregulated in the developing retina of mice and can promote differentiation of RPCs and gliogenesis in Müller cells. This study further revealed that let-7 may affect the self-renewal ability of RPCs by targeting the DNA structural protein HMGA2.18 Fumiko et al reported the roles of miR-9/9 and miR-124 in mouse retinal development. The results revealed that MiR-9/9 and miR-124 are highly expressed during retinal development. Overexpression of miR-9/9 and miR-124 in the neonatal retina promoted the neurogenesis of retinal ganglion cells, inhibited the gliogenesis of Müller cells and reduced the number of Sox9 (a marker of Müller cells) positive cells. This suggests that miR-9/9 and miR-124 may inhibit the expression of Sox9, which affects the Notch signalling pathway, thereby regulating gliogenesis in Müller cells.19 These results indicated the impact of miRNAs on Müller cell development and gliogenesis (figure 2).
Changes and roles of miRNA in Müller cells during retinal immunity and inflammation
Müller glial gliosis is an immune response to neural tissue damage, as indicated by high expression of GFAP.11 In a rat model of optic nerve crush injury, the intravitreal injection of a miR-21 inhibitor effectively suppressed Müller glial gliosis, promoted retinal ganglion cell survival and functional recovery, and increased the thickness of the retinal nerve fibre layer.20 However, the underlying mechanism remains unclear. In addition, the immune response to neural tissue damage leads to retinal inflammation. The selective deletion of Müller cells causes retinal inflammation and photoreceptor degeneration.21 Accompanying this change, the expression profiles of miRNAs and the retinal transcriptome are markedly altered.21 Twenty miRNAs and 78 target genes were altered after deletion, including Cyclin D2, Caspase 9, insulin-like growth factor 1, IL-1 receptor-associated kinase, calmodulin and Janus kinase 2. These genes also involve cellular apoptosis, p53, neurotrophin, calcium, chemokine and Jak-STAT signalling pathways.
During retinal injury or stimulation, the immune-related miRNA expression profile of Müller cells is significantly altered. Most miRNAs in mouse retinal Müller cells are downregulated after light injury, and only four miRNAs are upregulated, including miR-720, miR-29a, miR-29b, miR-124, and miR-1937a+b which regulate immune-related genes.22 Additionally, several changes in gene expression were observed. The top 10 expression-changed genes included Gfap, Serpina3n, Ednrb and Cxcl10.22 In addition, bioinformatics analysis showed changes in the expression of miR-125b-5p, let-7b and let-7c, and their target genes might be involved in response to stress and gliosis after light injury.22
The expression of miRNAs in Müller cells and their involvement in retinal inflammation has been reported in other retinal diseases. In a study on diabetic retinopathy, miR-365 expressed in Müller cells was found to target Timp3 and negatively regulate its expression, thereby activating Müller cells and promoting the expression of oxidative stress and retinal inflammatory factors.23 In human age-related macular degeneration and photooxidative damage in mouse retinas, the elevated expression of chemokine ligand 2 (Ccl2) is negatively correlated with miR-124 expression. Overexpression of miR-124 in the Müller cell line and mouse retina effectively downregulated the expression of Ccl2 and inflammatory factors.24 Furthermore, the overexpression of miR-124 in the mouse retina reduces the number of microglia and apoptotic photoreceptors and promotes the recovery of visual function.24
Kaur et al summarised the role of miRNAs in diabetic retinopathy and found that some miRNAs, such as miR-152 and miR-146, are involved in retinal inflammation in diabetic retinopathy.25 26 We also hypothesised that these miRNAs may affect Müller cell function. These studies show that miRNA-mediated Müller cell dysfunction plays a major role in immunity against retinal diseases (figure 3) and may be a potential therapeutic target for treating retinal diseases.
Roles of miRNA in Müller cells on retinal regeneration
Müller cells are the main neuroglia in the retina and can be used for retinal neuron regeneration. Several studies have reported the ability of miRNAs to regulate Müller cell reprogramming. In the retina of zebrafish, Müller cells proliferate easily and transform into neurons, known as the neural stem potential of neuroglia cells.7 However, in most mammals, retinal Müller cells are not easily converted into neural stem-like cells and can only be converted into neurons to repair tissue damage under injury conditions.7
Previous studies have reported the roles of miRNAs in Müller cell reprogramming in zebrafish. For example, miR-28 and miR-216a are involved in reprogramming Müller cells. Studies using cell models have revealed that miR-28 in Müller cells can target the photoreceptor-specific transcription factor CRX, inhibiting Müller cells from transforming into PRCs which further differentiate into photoreceptors.27 In zebrafish retinas, miR-216a inhibits the dedifferentiation and proliferation of Müller cells during retinal regeneration and sustains a strong light injury, leading to the downregulation of miR-216a in Müller cells.28 Further studies on the underlying mechanisms suggested that miR-216a targeted H3K79 methyl transferase Dotl, which was upregulated in proliferative Müller cells after retinal injury and regulated Wnt/β- Catenin pathway to promote cell reprogramming.28 Another study in zebrafish indicated that the expression of the primitive neural transcription factor ascl1a and pluripotent factor Lin-28 in the retina was significantly upregulated after retinal injury, thus inducing Müller cells to acquire neural stem potential.29 Upregulation of Lin-28 expression inhibited the expression of let-7, which inhibited the expression of regeneration-related genes, such as ascl1a, hspd1, lin-28, oct4, pax6b, and c-Myc. Therefore, the expression of let-7 regulated by Lin-28 enhanced the neural stem potential of Müller cells.29 In addition, two miRNAs and genes (Tgf-β and Oct4) could effectively induce the reprogramming of Müller cells into RPCs in the damaged retina of zebrafish and could regulate a variety of regenerative transcription factors (ascl1a, lin28a, E-cadherin, sox2 and zebs) and miRNAs (miR-200a, miR-200b, miR-143 and miR-145).30 31
Studies have also reported a role for miRNAs in Müller cell reprogramming in injured mouse retinas. Regulation of let-7 expression by Wnt signalling and Lin28 affects the proliferation and conversion of Müller cells. Wnt-Lin28-let7 miRNA signal transduction plays a major role in regulating Müller cell proliferation in adult mammalian retinas and its potential for neurogenesis.32 Another study on mouse retinas reported that overexpression of miR-25 and miR-124 or antagonistic expression of let-7 induced elevated expression of ascl1 and transformed approximately 40% of mature Müller cells into neurones or RPCs.33 Furthermore, the target genes of these miRNAs were predicted, including the transcription factors Klf4, Dkk3, Tpm1, Itgb1 and Ctdsp1, which regulate the reprogramming of Müller cells mediated by ascl1 through the reporter element 1 silencing transcription factor (REST) pathway.33
Inflammation inhibits miRNA-mediated reprogramming of Müller cells. A study on the zebrafish retina reported that the expression of miR-18a increased after injury to the retinal photoreceptor cells. Müller cells were reprogrammed into photoreceptor cells to cope with the injury. However, after mutation of miR-18a in the retina, retinal microglia are activated, the expression of inflammatory genes is upregulated, and the conversion of Müller cells into neurones is inhibited.34
These results indicate that miRNAs participate in reprogramming Müller cells and mediate retinal regeneration (figure 4), which may provide novel insights into treating retinal injury and repair in the clinic.
Roles of miRNA in Müller cells during the progress of retinal disorders
MiRNAs participate in the progression of retinal diseases via Müller cells. In glaucoma models, changes in the expression of miR-200a35 and miR-61536 led to alterations in the function and survival of retinal ganglion cells and were accompanied by changes in Müller cell function, suggesting the possible participation of miRNAs in the development of glaucoma through Müller cells. Direct involvement of miRNA in retinal disorders showed that Müller cells derived miR-124 promoted the growth of axons of retinal ganglion cells in the glaucoma model. Moreover, miR-124 promotes the growth of neurites and guides retinal ganglion cells by targeting and downregulating the cofactor of REST to promote the expression of Neurobilin-1 and counter disease progression.37
In diabetes retinas, miRNA-15-5 p, 27b-3p, and 107-3p are highly expressed, and they target aquaporin 11, inhibit its expression and cause Müller cell oedema, thereby promoting disease progression of the disease.38 A similar study showed that glioma-associated oncogene homologue 1 in the retina of diabetic individuals regulates the expression of miR-29 in Müller cells. This leads to the regulation of the expression of the target gene forkhead box protein O4 of miR-29, causing Müller cell dysfunction and exacerbating disease progression.39 In another study, the expression of miR-497 in Müller cells negatively regulated brain-derived neurotrophic factors promoted apoptosis and exacerbated the progression of diabetic retinopathy.40 Finally, the study found that downregulation of miR-29b expression in the diabetic retina can effectively promote the transcription of its target gene, specificity protein 1, thereby inhibiting the apoptosis of Müller cells and preventing disease progression.41 Therefore, miRNA-mediated Müller cells may serve as therapeutic targets in retinal diseases.
Roles of miRNA in Müller cells on retinal development
Although there are few reports on the miRNA-mediated participation of Müller cells in retinal development, some studies have shown that miRNAs in Müller cells are key elements in maintaining retinal structure and stability. Wohl et al reported the migration of many Müller cells and their aggregation after knocking out Dicer 1 in mouse Müller cells, seriously damaging normal retinal structure and function.42 After Dicer 1 knockout, 23 miRNAs that were originally highly expressed in Müller cells were downregulated, including miR-204, miR-125b-5p, miR-9, miR-181a, miR-99a, let-7c, let-7b, miR-720, miR-30c and others. The upregulation of brevican protein expression after Dicer 1 knockout is the main reason for the migration and aggregation of many Müller cells.42 Further studies indicated that brevican is the target gene of miR-9. Overexpression of miR-9 in Müller cells knocked out by Dicer 1 effectively reversed the abnormal migration of Müller cells.42 Therefore, miRNA-mediated Müller cells may be involved in retinal development.
In this review, the roles of miRNA-mediated Müller cells in retinal function, including gliogenesis, inflammation and immunity, regeneration, development of retinal diseases, and retinal development, are described and summarised in figure 5. The categories of miRNAs and their target molecules were analysed and are listed in table 1. The possible mechanisms and signalling pathways of the target molecules in each function are also discussed. In addition, because miRNAs are short nucleotide sequences, they are easier to modify or detect in exosomes. These identified miRNAs may be used as potential biomarkers for diagnosing retinal diseases and as treatment targets in the future.
Data availability statement
The data sets used and analysed during the current study are included within the article and its additional files. All material used in this review are from publicly available articles.
Patient consent for publication
WJ and SH contributed equally.
Contributors JZ, QX, LQ and YX contributed to conceptualisation. JZ, LQ and QX contributed to investigation and supervision. JZ, WJ, SH, LL, XM, JLu, JLi and TC were involved in visualisation, literature search and writing—original draft, and JZ, WJ, SH, LL XM, JLu, TC QX, LQ and YX contributed to writing—review and editing.
Funding This work was supported by Guangdong Basic and Applied Basic Research Foundation (grant number 2020A1515110421), National Natural Science Foundation of China (grant number 82101165), Natural Science Foundation of Hunan Province (grant number 2022JJ30522).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.