MicroRNA-29b Overexpression Decreases Extracellular Matrix mRNA and Protein Production in Human Corneal Endothelial Cells

Toyono, Tetsuya; Usui, Tomohiro; Villarreal, Guadalupe; Kallay, Laura; Matthaei, Mario; Vianna, Lucas Monferrari Monteiro; Zhu, Angela Y.; Kuroda, Masahiko; Amano, Shiro; Jun, Albert S.

doi:10.1097/ico.0000000000000954

Cited by 36 publications

(22 citation statements)

References 33 publications

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“…We performed a GO-term enrichment analysis of miR-29 targets with 8-mer seed matches and found that about half of the top pathways were related to ECM structure or insulin signaling, consistent with miR-29’s previously described roles in liver, cornea, muscle, and heart (Supplementary Fig. 2c) 21,22,23,24 . A closer examination of individual miR-29 targets, ranked by peak height, reveals that 4 of the top 10 genes have been previously validated in other cell types (Fig.…”

Section: Figsupporting

confidence: 81%

AGO HITS-CLIP in Adipose Tissue Reveals miR-29 as a Post-Transcriptional Regulator of Leptin

O’Connor

Murphy

et al. 2020

Preprint

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MicroRNAs (miRNAs) are short, non-coding RNAs that associate with Argonaute (AGO) to regulate mRNA stability and translation. While individual miRNAs have been shown to play important roles in white and brown adipose tissue in normal physiology and disease 1,2,3 , a comprehensive analysis of miRNA activity in these tissues has not been performed. We used high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) to comprehensively characterize the network of high-confidence, in vivo mRNA:miRNA interactions across white and brown fat, revealing over 100,000 unique miRNA binding sites. Targets for each miRNA were ranked to generate a catalog of miRNA binding activity, and the miR-29 family emerged as a top regulator of adipose tissue gene expression. Among the top targets of miR-29 was leptin, an adipocyte-derived hormone that acts on the brain to regulate food intake and energy expenditure 4 . Two independent miR-29 binding sites in the leptin 3'-UTR were validated using luciferase assays, and miR-29 gain and loss-of-function modulated leptin mRNA and protein secretion in primary adipocytes. In mice, miR-29 abundance inversely correlated with leptin levels in two independent models of obesity. This work represents the only experimentally generated miRNA targetome in adipose tissue and identifies the first known post-transcriptional regulator of leptin. Future work aimed at manipulating miR-29:leptin binding may provide a therapeutic opportunity to treat obesity and its sequelae.Much of the work on adipocyte development and function over the last several decades has focused on transcriptional regulators. These include PPARG2, which is necessary and sufficient for the development and maintenance of white and brown adipocytes 5 , and PRDM16, which regulates brown and beige adipocyte identity 6,7 . More recently, it has become clear that post-transcriptional regulators also play 3 a key role in influencing adipocyte phenotype. Studies with adipose-specific dicer KO mice demonstrate that microRNAs (miRNAs) are essential in regulating adipogenesis, insulin sensitivity and non-shivering thermogenesis 8,9 . Several individual miRNAs have been shown to contribute to these phenotypic effects.miR-133 represses BAT function by directly targeting Prdm16 and inhibiting expression of thermogenic genes 2 . miR-196a induces browning of WAT by targeting Hoxc8 1 , and miR-26 protects mice from dietinduced obesity by blocking adipogenesis 10 . These studies, and most others in adipose tissue, rely on computational predictions and low-throughput validation to identify miRNA targets. Although highthroughput crosslinking techniques to map the miRNA targetome have been applied to liver, brain, heart and other tissues 11,12,13 , thus far, no comprehensive targetome has been described for adipose tissue.To profile the complete network of in vivo, adipose-specific miRNA binding activity across both brown and white fat, we used AGO HITS-CLIP on interscapular brown adipose tissue (iBAT) and epididymal white a...

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Section: Figsupporting

confidence: 81%

AGO HITS-CLIP in Adipose Tissue Reveals miR-29 as a Post-Transcriptional Regulator of Leptin

O’Connor

Murphy

et al. 2020

Preprint

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“…Recent studies have shown that miRNAs-29a/b/c were involved in osteoclast regulation and could influence expression of certain extracellular matrix molecules i.e. collagens and their modifying enzymes[ 13 – 17 ]. Previous reports showed an increase in miRNA-29b during osteoclast differentiation in TNF- α / RANKL-treated cells[ 16 ] and promoted osteoclastogenesis by regulating osteoclast commitment and migration[ 17 ].…”

Section: Discussionmentioning

confidence: 99%

“…miRNAs-29a/b/c were involved in regulation of osteoblasts/clasts differentiation and could influence expression of certain extracellular matrix molecules i.e. collagens and their modifying enzymes[ 13 – 15 ]. Kagiya and Nakamura[ 16 ] found an increase in miRNA-29b during osteoclast differentiation in TNF-α/RANKL-treated cells, suggesting this miRNA plays a role in TNFα -regulated osteoclast differentiation.…”

Section: Introductionmentioning

confidence: 99%

Secretory microRNA-29 expression in gingival crevicular fluid during orthodontic tooth movement

et al. 2018

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Secretory microRNAs (miRNAs) have been used increasingly as biomarkers for cancers, autoimmune diseases and inflammatory diseases. They are reported as being freely circulated or encapsulated in microvesicles such as exosomes. This study was performed to elucidate the presence of miRNAs with exosomes in human gingival crevicular fluid (GCF), and the expression profile of miRNA-29 during orthodontic tooth movement. Four healthy volunteer and fifteen orthodontic patients were enrolled in the study. Secretory miRNA in GCF was collected and analyzed using a bioanalyzer, realtime PCR and Western blot analysis. The expression profile of secretory miR-29 family in GCF was analyzed during the course of canine retraction for 6 weeks. The results demonstrated the presence of miRNAs in the GCF. After series of ultracentrifugation and RT-PCR array, exosome-depleted fractions and pellets were isolated and we found that secretory miRNAs were detected in both the exosome-associated fraction and the exosome-depleted supernatant fraction; however, the concentration of miRNAs was higher in the exosome-associated fraction than in the exosome-depleted fraction suggesting a close association between the secretory miRNAs and exosomes in GCF. We also demonstrated the increased expression profiles of miR-29 family during six weeks of orthodontic tooth movement in humans. Secretory miRNAs are present in GCF and secretory miRNA-29 family expression profiles increase during the tooth movement in humans. Secretory miRNA-29 in GCF could serve as potential biomarkers for periodontal remodeling.

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“…We previously identified global DNA methylation changes that occur in the corneal endothelial tissue of FECD patients and specifically observed a high number of DNA methylation alterations occurring in miRNA sequences [30]. This finding was intriguing because prior reports had demonstrated that miRNAs were differentially expressed in the corneal endothelium during aging [58], and that widespread downregulation of miRNA levels occurred in the corneal endothelium of patients with late-onset FECD [36, 59]. Because DNA methylation has been shown to be a mechanism for regulating miRNA expression [33], we performed a sub-analysis of the miRNA DNA methylation array data.…”

Section: Discussionmentioning

confidence: 99%

Aberrant DNA methylation of miRNAs in Fuchs endothelial corneal dystrophy

Pan

Weisenberger

Zheng

et al. 2019

Preprint

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18Homeostatic maintenance of corneal endothelial cells is essential for maintenance of corneal 19 deturgescence and corneal transparency. In Fuchs endothelial corneal dystrophy (FECD), an 20 accelerated loss and dysfunction of endothelial cells leads to progressively severe visual 21impairment. An abnormal accumulation of extracellular matrix is a distinctive hallmark of the 22 disease, however the molecular pathogenic mechanisms underlying this phenomenon are not fully 23 understood. We recently reported characteristic patterns of DNA methylation changes in the 24 corneal endothelial cells of patients with FECD. Here, we investigate genome-wide and sequence-25 specific DNA methylation changes of miRNA genes in corneal endothelial samples derived from 26 patients with FECD. We show that the majority of miRNA genes are hypermethylated at their 27 promoter regions in FECD. More specifically, miR-199B is an extensively hypermethylated 28 miRNA gene at its promoter region and its mature transcript miR-199b-5p was previously found 29 to be almost completely silenced in FECD. Using a cell-based assay, we find that miR-199b-5p 30 directly inhibits the expression of two epithelial mesenchymal transition (EMT)-inducing genes, 31Snai1 and ZEB1. Taken together, these findings suggest a novel regulatory mechanism of matrix 32 protein production by corneal endothelial cells in which miR-199b-5p hypermethylation leads to 33 its down-regulated expression and thereby the decreased expression of miR-199b-5p target genes, 34including Snai1 and ZEB1. Our results support miR-199b-5p as a potential therapeutic target to 35 prevent or slow down the progression of FECD disease. 36 37 Author summary 38 Fuchs endothelial corneal dystrophy (FECD) due to corneal endothelial cell degeneration is one 39 of the most common heritable causes of corneal visual loss and a leading indication for corneal 40 3 transplantation. The progressive loss of corneal endothelial cells is accompanied by an abnormal 41 deposition of extracellular matrix in the form of guttae. Here we discover that miRNA gene 42 promoters are frequent targets of aberrant DNA methylation in FECD. In particular, we describe 43 a novel epigenetic mechanism used by corneal endothelial cells to regulate extracellular matrix 44 production. We find that miRNA-199b-5p functions as a negative regulator of Snai1 and ZEB1, 45 two zinc finger transcription factors that have been shown to lead to increased production of 46 extracellular matrix proteins. Furthermore, miR-199B was extensively hypermethylated in FECD 47 and its mature transcript miR-199b-5p directly binds to the 3′-UTRs of Snai1 and ZEB1 genes. 48Ultimately, this may negatively modulate Snai1-and ZEB1-mediated production of extracellular 49 matrix proteins. This work is the first to identify an important role of DNA methylation in the 50 epigenetic regulation of miRNA-target genes in FECD and to describe a potential epigenetic 51 biomarker for the treatment of FECD patients. 52 53

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MicroRNA-29b Overexpression Decreases Extracellular Matrix mRNA and Protein Production in Human Corneal Endothelial Cells

Cited by 36 publications

References 33 publications

AGO HITS-CLIP in Adipose Tissue Reveals miR-29 as a Post-Transcriptional Regulator of Leptin

AGO HITS-CLIP in Adipose Tissue Reveals miR-29 as a Post-Transcriptional Regulator of Leptin

Secretory microRNA-29 expression in gingival crevicular fluid during orthodontic tooth movement

Aberrant DNA methylation of miRNAs in Fuchs endothelial corneal dystrophy

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