*6.3. Chromatin Remodeling*

Chromatin remodeling is the conjunction of various structural and/or molecular changes that ultimately modify DNA functions as expression, replication and recombination of genes. Therefore, without having genomic alterations the production of proteins will be a ffected and may be inherited to o ffspring [258].

Histone modifiers (acetylation, deacetylation, methylation, and demethylation enzymes), histone chaperons and ATP-dependent chromatin remodelers are responsible for chromatin restructuring [263]. Four families of chromatin remodelers have been described: SWI/SNF, NO80, CHD and ISWI. This very last is associated to the Base Excision Repair (BER) and the Nucleotide Excision Repair (NER) mechanisms to help repair DNA after an oxidative insult [264]. Chromatin remodeling by its remodeler family SWI/SNF contributes to the response against stress and senescence, at the same time mitochondrial dysfunction induces chromatin remodeling responses [265]. The thioredoxing-interacting protein (TXNIP) is a protein known to have an important impact over OS, inflammation and apoptosis in the pancreas and the retina [266], in recent years it has been considered that these modifications lead to chromatin remodeling in retinal cells. During hyperglycemia some molecules such as Angiotensin II, transforming growth factor-β (TGF-β) and AGEs are overexpressed leading to enhanced activation of their receptors causing augmented production or intracellular factors that end up modifying chromatin structure [267].

The DNA binding proteins HMGBs (high mobility group B) can mediate nucleosome remodeling. Such proteins lower their a ffinity to DNA and their function when oxidized [268]. OS promotes alterations in chromatin by histone acetylation and increased activation of NF-kB resulting in major inflammatory genes expression [269]. Second messengers are also able to modify chromatin structure via chromatin-binding proteins. PI5P (phosphoinositide phosphatidylinositol-5-phosphate) is an example, its production augments in OS environment, it binds to ING2 (inhibitor of growth family member 2) to repress pro-proliferative genes and promote apoptosis [270,271].

## *6.4. Non-Coding RNA*

Non-Coding RNAs are RNAs that are not translated into proteins, and instead they play a role as gene expression modulators. There are two types of non-coding RNAs classified by their length, small RNAs formed by 20–30 nucleotides and LncRNAs (long non-coding RNAs) having up to 200 nucleotides [272]. Small non-coding RNAs (20–30 nucleotides) are categorized as microRNAs (miRNAs), short interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs). The first two are more similar between each other as they both have double-stranded precursors, while piRNAs appear to derive from single-stranded precursors [273].

MiRNAs have a regulatory function on endogenous genes, and siRNAs protect the genome from invasive nucleic acids [273]. Both molecules work primarily by silencing genes recognized by a specific sequence in the miRNA or siRNA chain. Normally, miRNAs inhibit the expression of their target genes promoting its mRNAs degradation or inhibition of its translation [274], but this programmed action may be modified in response to external threats (viruses, transposons) or changes within the cell. Then, siRNAs will co-opt the invader into their own mechanism and prevent it from its expression; on the other hand, miRNAs may be diluted or exchanged by different miRNAs or even new miRNAs will be expressed to silence genes that will have the effect of counteracting the previous silencing [273,275]. Most lncRNAs are transcribed by RNA polymerase II and spliced into various isoforms which act as epigenetic regulators. They can regulate DNA polymerase activity, around 20% of lncRNAs recruit chromatin remodeling complexes to repress transcription of target genes [276,277].

Through the years non-coding RNAs have been related to many degenerative illnesses, including Alzheimer, cancer and diabetes [272,274,277]. As mentioned before, when cells encounter environmental modifications, such as hyperglycemia, they promote altered expression of miRNAs. The downregulation of certain miRNAs (miR-126 and miR-200b) and the upregulation of others (miR-18a, miR-20b, miR31 an mir-155) are now related to augmented VEGF production [278], miR-146 is a miRNA that functions as a negative feedback to NF-kB activation induced by IL-1β in retinal endothelial cells and it is also upregulated after 3 months following the onset of diabetes [278].

Some miRNas such as miR27b respond to stress by reducing the expression of pro-antioxidant proteins as Nrf2 resulting in a pro-oxidant environment [279]. mR27b also has a pro-angiogenic effect targeting Notch ligand D114, Sprouty -2, PPARgamma and Semaphorin 6A [280]. Another miRNA; miR-211 is upregulated in diabetes causing impaired expression of sirtuin-1 [281], a protein that protects mitochondria from damage [282]. In the past years over 47 miRNAs have been found differential between patients with diabetes and patients with DR, five of them, including miR-21 implicated in angiogenic processes, have been considered as biomarkers for early detection of such microvascular complication [283,284].

miRNAs associate with groups of proteins known as Argonaute proteins to silence the target gene. Argonaute proteins are usually found scattered in the cytoplasm, in oxidative stress conditions they tend to gather around stress granules, an action mediated by miRNAs [285]. On the other hand, argonaute2 is enhanced by hypoxia which leads to an inhibited maturation of miRNAs that respond to stress as defense mechanisms [286]. Previously mentioned miRNAs have been found in serum, some of them and others are found in vitreous humor and aqueous humor in PDR as seen in Table 2. miR-126 has recently been considered as a potential serum biomarker for PDR nonetheless, other studies have shown that the concentration of miRNAs in eye matrices with PDR differs even two times fold from those in serum [287].

H-19, a lncRNA that prevents glucose-induced endothelial mesenchymal transition in the retina, is downregulated in hyperglycemic conditions leading to activation of endothelial transition via TFGF-beta [288]. A recent study has found that lncRNA AK077216 is downregulated in subjects with DR independently from glycemic conditions, this lncRNA is able to inhibit ARPE-19 cells apoptosis via inhibition of miR-383 proapototic function [289]. The proliferation promoting lncRNA BANCR has also been found overexpressed in patients with DR which may play a role as a potential therapeutic target [290,291]. According to Awata and cols. there is an association between susceptibility to DR and lncRNA RP1-90L14 [91].

Studies have shown that these alterations occur early in hyperglycemic states in retinal and endothelial cells [292], furthermore; they are maintained even after returning to normoglycemia [293–295]. Since non-coding RNA, specially miRNAs have a regulatory function, relatively prompt response to alterations in the body and, more importantly, such responses are maintained for a period of time after glycemic control they represent a potential therapeutic target in DR to delay its progression. Nevertheless more studies are needed so far.
