Abstract
During development, tight regulation of the expansion of retinal progenitor cells (RPCs) and their differentiation into neuronal and glial cells is important for retinal formation and function. Our study demonstrated that microRNA (miR)-29a modulated the proliferation and differentiation of RPCs by suppressing RBM8A (one of the factors in the exon junction complex). Particularly, overexpression of miR-29a reduced RPC proliferation but accelerated RPC differentiation. By contrast, reduction of endogenous miR-29a elicited the opposite effects. Overexpression of miR-29a repressed the translation of Rbm8a, thus negatively regulating RPC proliferation and promoting the neuronal and glial differentiation of RPCs, and knockdown of endogenous Rbm8a phenocopied the observed effects of miR-29a overexpression. Furthermore, a luciferase reporter assay showed that miR-29a directly interacted with the Rbm8a mRNA 3′UTR, which indicated that Rbm8a is the direct target of miR-29a. To further verify the result, co-overexpression of the Rbm8a 3′ UTR-wt (plasmids into which the Rbm8a 3′ UTR sequence had been introduced) and miR-29a in RPCs rescued the phenotype associated with miR-29a overexpression, reversing the promotion of differentiation and inhibition of proliferation. These results show a novel mechanism by which miR-29a regulates the proliferation and differentiation of RPCs through Rbm8a.
Highlights
Retinal progenitor cells (RPCs) are a subset of undifferentiated cells with indefinite potential for selfrenewal and differentiation into retinal neuronal and glial lineages [1, 2]
To reveal the function of miR-29a in retinal progenitor cells (RPCs), our study first investigated the expression of mi-29a during RPC differentiation. qRT-PCR results showed that miR-29a expression significantly increased after induction of RPC differentiation (RPCs cultured in differentiation medium), reaching an approximately 4-fold peak at day 7, compared with undifferentiated cells (RPCs cultured in proliferation medium) (Figure 1A)
QPCR detected that the expression of proliferative marker (Ki67), and retinal progenitor markers was gradual decreased, but the expression of RPC differentiation markers (Rhodopsin, a marker for photoreceptors; β3-tubulin, a pan-neuronal marker; PKC-α, a marker for bipolar neurons, and glial fibrillary acidic protein (GFAP), a glial cell marker) had the reversed trend during RPC differentiation and it showed that RPCs were gradually differential over time (Figure 1F and 1G)
Summary
Retinal progenitor cells (RPCs) are a subset of undifferentiated cells with indefinite potential for selfrenewal and differentiation into retinal neuronal and glial lineages [1, 2]. Retinal neurons cannot self-repair after injury or exposure to pathological conditions [6, 7], emphasizing the importance of better understanding the mechanisms of controlling RPC differentiation. RPC self-renewal and differentiation is tightly regulated by a series of events including dynamic changes in the pattern of expression of microRNAs (miRNAs) that drive cellular fate determination [8, 9]. MiR-29a has been shown to be expressed in mesenchymal stem cells and to serve as an important regulator in the neuronal differentiation of mesenchymal stem cells by targeting REST [18]. A study detected that miR-29a expression changes through Müller glia dedifferentiation and CD73+ rod photoreceptor differentiation [19]. Whether miR-29a regulates the proliferation and differentiation of RPCs remains unknown
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