Synchronous down-modulation of miR-17 family members is an early causative event in the retinal angiogenic switch

Abstract

Six members of the microRNA-17 (miR-17) family were mapped to three different chromosomes, although they share the same seed sequence and are predicted to target common genes, among which are those encoding hypoxia-inducible factor-1α (HIF1A) and VEGFA. Here, we evaluated the in vivo expression profile of the miR-17 family in the murine retinopathy of prematurity (ROP) model, whereby Vegfa expression is highly enhanced at the early stage of retinal neovascularization, and we found simultaneous reduction of all miR-17 family members at this stage. Using gene reporter assays, we observed binding of these miRs to specific sites in the 3′ UTRs of Hif1a and Vegfa. Furthermore, overexpression of these miRs decreased HIF1A and VEGFA expression in vitro. Our data indicate that this miR-17 family elicits a regulatory synergistic down-regulation of Hif1a and Vegfa expression in this biological model. We propose the existence of a coordinated regulatory network, in which diverse miRs are synchronously regulated to target the Hif1a transcription factor, which in turn, potentiates and reinforces the regulatory effects of the miRs on Vegfa to trigger and sustain a significant physiological response. In humans, the miR-17 family is composed of six distinct mature miRs located on three chromosomes: miR-17-5p and miR-20a are located on chromosome 13q31.3, miR-20b and miR106a are located on chromosome Xq26.2, and miR-106b and miR-93 are located on chromosome 7q22.1. Members of this family seem to be derived from gene duplication events (9), and despite some divergences in length and nucleotide composition, their seed sequence (AAAGUG) is identical, an attribute suggestive of functional redundancy (10). Although the miR-17-92 cluster has been well-characterized, the regulatory role of the miR-17 family members has not been extensively studied (11,12). Distinct prediction algorithms (13-16) indicate that the miR-17 miR family may target the 3′ UTRs of genes encoding hypoxia-inducible factor-1α (HIF1A) and VEGFA. Therefore, by simultaneously targeting these two key genes, miR-17 family members could, in theory, be important concerted regulators of angiogenesis. As proof of concept, we tested this hypothesis in the experimental mouse model of retinopathy of prematurity (ROP), where Vegfa seems to be a pivotal factor that modulates the angiogenic switch (17)(18)(19). In this model, the expression of retinal Vegfa mRNA increases sharply 12 h after the animals are returned from 75% oxygen (O 2 ) back to room air (∼21% O 2 ), a process that induces a relative hypoxic condition and leads to retinal neovascularization in the subsequent 9 d (17-19).Here, we show the synchronous down-regulation of all members of the miR-17 family in the critical early steps of neovascularization in the ROP model. We propose an miR regulatory network, in which distinct and partially redundant miR-17 family members simultaneously affect the levels of the Hif1a transcription factor to posttranscriptionally (either directly and/or indirectly) increase the expres...

Retinal and circulating miRNA expression patterns in diabetic retinopathy: An in silico and in vivo approach

“…Four miRNAs were found to be downregulated in retina and serum of diabetic mice: miR-20a-5p, miR-20a-3p, miR-20b, and miR-106a-5p. These data are in agreement with a previous report about the role of miR-20a-5p, miR-20a-3p, miR-20b, and miR-106a-5p on regulation, at the post-transcriptional level, of genes involved in angiogenesis (Nunes et al, 2015). Our data indicated (Nunes et al, 2015).…”

“…These data are in agreement with a previous report about the role of miR-20a-5p, miR-20a-3p, miR-20b, and miR-106a-5p on regulation, at the post-transcriptional level, of genes involved in angiogenesis (Nunes et al, 2015). Our data indicated (Nunes et al, 2015). Additionally, Nunes and co-authors validated VEGF and HIF as gene targets of these four miRNAs, that, when up-regulated, promote degradation of VEGF and HIF mRNA.…”

“…Our data indicated that early diabetes led to down‐regulation of miR‐20a‐5p, miR‐20a‐3p, miR‐20b, and miR‐106a‐5p and therefore to an angiogenic switch in the retina. Nunes et al's paper reported that down‐regulation of miR‐20a‐5p, miR‐20a‐3p, miR‐20b, and miR‐106a‐5p caused retinal neovascularization in vivo (Nunes et al, ). Additionally, Nunes and co‐authors validated VEGF and HIF as gene targets of these four miRNAs, that, when up‐regulated, promote degradation of VEGF and HIF mRNA.…”

“…Top scored miRNAs, to be further analysed in the in vivo models, were selected as the nodes bearing the highest closeness centrality and degree values (Koschutzki & Schreiber, 2008), because high closeness centrality or high degree nodes are the most connected miRNAs in the miRNA-pathway network. Analysis was integrated with literature search results ( Figure S1; Nunes et al, 2015;Qin, Liu, Liu, Li, & Ren, 2016;Zampetaki et al, 2016).…”

Resolvin D1 Modulates the Intracellular VEGF-Related miRNAs of Retinal Photoreceptors Challenged With High Glucose

“…Among these increased VEGF and decrease of related inhibitory miRNAs are demonstrated to result in cells damage and modified angiogenesis in diabetic retinopathy (He and King, 2004;Kowluru and Chan, 2007;Patel and Santani, 2009;Kitada et al, 2010;Szade et al, 2015;Suryavanshi and Kulkarni, 2017;Yin et al, 2017;Platania et al, 2019). microRNAs (miRNAs), small non-coding RNA molecules that regulate gene expression at post-transcriptional level, have recently emerged as essential regulators for the orchestration of retina homeostasis and angiogenesis (Nunes et al, 2015;Platania et al, 2019). Although we do not know which ones are more important at mediating the effects or whether their expression is modulated differentially, here it is depicted a down-regulation of four intracellular VEGF-related inhibitory miRNAs involved in the control of neovasculature.…”