Implications of long non-coding RNAs in the pathogenesis of diabetic retinopathy: a novel epigenetic paradigm.
Abstract
With the rising incidence of diabetic retinopathy (DR), there is an urgent need for novel therapies. Presently, several altered metabolic pathways have been implicated in the pathogenesis of DR. Recent advances in genomic technologies have identified considerable epigenetic alterations that also contribute to DR progression. Long non-coding RNAs (lncRNAs; >200 nucleotides), critical regulators of gene expression, are aberrantly expressed in DR and have not been comprehensively characterized. Our microarray analyses using human retinal endothelial cells (HRECs) revealed thousands of differentially expressed lncRNAs following high glucose (HG) exposure, with profound increases in the lncRNAs MALAT1 and HOTAIR. Using multiple techniques, I sought to elucidate the roles of these two molecules in inflammation and angiogenesis during DR. My findings demonstrated that MALAT1 is upregulated in HG and in diabetic animals, and regulates inflammatory transcripts (IL-6 and TNF-α) through its association with polycomb repressive complex 2 (PRC2). Vitreous humors from diabetic patients revealed parallel findings. DNA methylation array analyses did not demonstrate significant alterations at CpG sites across the MALAT1 gene, but inhibition of DNA methyltransferases significantly increased MALAT1 and associated inflammatory transcripts. Furthermore, HG upregulated HOTAIR and angiogenic transcripts (VEGF-A and ET-1) in HRECs and promoted an association with RNA-binding proteins, P300 and EZH2. HOTAIR knockdown reduced the expressions of angiogenic cytokines, EZH2 and P300. HG did not induce significant hypomethylation in HOTAIR CpG regions, while inhibitors for histone methylation, DNA methylation and HOTAIR significantly impacted VEGF-A and ET-1 expressions. HOTAIR expressions were elevated in the vitreous of DR patients and in the retinas of diabetic rodents. HOTAIR knockdown reduced HG-induced oxidative DNA and mitochondrial damage. The studies were further extended to delineate how these epigenetic mechanisms influence the regulation of a specific vasoactive factor, ET-1, in DR. DNA methylation array demonstrated hypomethylation in the ET1 promoter in HG. Blocking DNA methylation or histone methylation significantly increased ET-1 mRNA expressions in control and HG-treated HRECs; while, knocking down pathogenetic lncRNAs (MALAT1 and HOTAIR) subsequently prevented glucose-induced ET-1 upregulation. Collectively, I uncovered a novel epigenetic paradigm that demonstrates a complex web of epigenetic mechanisms that regulate glucose-induced transcription of molecules in important pathological processes (inflammation and angiogenesis) during DR.