Nerve Growth Factor (NGF) is a member of the neurotrophins family that plays a key role in the maintenance and functions of both the central and peripheral nervous systems, as well as in non-neuronal cells. It exerts its action through two classes of transmembrane receptors, one high affinity, TrkA, and one low affinity, p75NTR, regulating different and important biological functions. Besides several neuropathies, some studies showed that NGF revealed translational clinical application for ulcers of the eye’s anterior segment, and a NGF bioformulation has been EMA and FDA approved as first-in-class treatment for neurotrophic keratitis (NK), a rare degenerative corneal disease due to corneal trigeminal innervation defects causing spontaneous injuries and wounds. However, NGF-induced biomolecular mechanisms in corneal cells are still unknown. MicroRNAs (or miRNAs) are a novel class of endogenous, small non-coding RNAs (~22 nucleotides), able to regulate target genes, at the post-transcriptional level, in plants and animals. MiRNAs are involved in maintaining cell homeostasis and tuning fundamental biological processes, such as growth, proliferation, apoptosis, cell metabolism; dysregulated expression of miRNAs’ is observed and strictly linked to relevant human diseases (e.g. viral, immune, neurodegenerative and cardiovascular diseases, cancer). In this study we evaluated miRNAs’ expression modulation in corneal cells after treatment with different NGF bioformulations, by focusing the analysis on target genes and pathways as well. Tens of miRNAs were significantly dysregulated, mainly hypo-expressed, after treatment. Among those, 3 were shared by all the bioformulations, 37 by 2 of them, whereas 12, 16 and 20 were correlated to each single bioformulation. A total number of 91 unique pathways were identified by DIANA experimentally-supported Tarbase analysis: 46, including the neurotrophin signalling pathway, were shared by all three bioformulations, 28 by 2 of them, whereas 9, 2 and 6 were related to each single bioformulation. More than 2.000 experimentally supported target genes were identified. After focusing the analysis on the neurotrophin signalling pathway, no relevant differences were globally detected among the NGF bioformulations, all appearing to drive through proliferative and survival signals. However, one of the three bioformulations here used appeared to be more specific than the remaining two, as, when proportionally compared to them, it involved less significant miRNAs which, however, can target higher numbers of genes involved in this pathway. The same result was achieved when analysis was restricted to miRNAs’ sub-groups specifically related to each single bioformulation. Genemania gene and functional enrichment analysis further confirmed data. Futhermore, the gene list can be in depth analysed in order to identify targets suitable for further molecular analyses and validations, especially for each single bioformulation, with the aim to possibly identify target genes and functions specifically related to them. For this reason, more experiments by immunoblotting to evaluate expression levels of interesting target genes are ongoing. We examined the effects induced by different NGF bioformulations on epithelial corneal cells, by focusing the attention on miRNAs expression levels, and in silico target genes and pathways analysis. Although further functional studies to validate results are needed, original and novel insights about genes and epigenetics mechanisms induced by NGFs were provided. Our data may leave to hypothesize that NGF1 might be more specific and effective in inducing neurotrophin signalling pathway compared with NGF2 and NGF3. Importantly, given the putative role of miRNAs as biomarkers or therapeutic targets, this study makes available data potentially exploitable in clinical practice.

Studio dell'espressione di microRNA, geni target e pathways indotti da NGF in cellule epiteliali di cornea: confronto tra differenti bioformulazioni / Compagnoni, Chiara. - (2020 Jul 07).

Studio dell'espressione di microRNA, geni target e pathways indotti da NGF in cellule epiteliali di cornea: confronto tra differenti bioformulazioni.

COMPAGNONI, CHIARA
2020

Abstract

Nerve Growth Factor (NGF) is a member of the neurotrophins family that plays a key role in the maintenance and functions of both the central and peripheral nervous systems, as well as in non-neuronal cells. It exerts its action through two classes of transmembrane receptors, one high affinity, TrkA, and one low affinity, p75NTR, regulating different and important biological functions. Besides several neuropathies, some studies showed that NGF revealed translational clinical application for ulcers of the eye’s anterior segment, and a NGF bioformulation has been EMA and FDA approved as first-in-class treatment for neurotrophic keratitis (NK), a rare degenerative corneal disease due to corneal trigeminal innervation defects causing spontaneous injuries and wounds. However, NGF-induced biomolecular mechanisms in corneal cells are still unknown. MicroRNAs (or miRNAs) are a novel class of endogenous, small non-coding RNAs (~22 nucleotides), able to regulate target genes, at the post-transcriptional level, in plants and animals. MiRNAs are involved in maintaining cell homeostasis and tuning fundamental biological processes, such as growth, proliferation, apoptosis, cell metabolism; dysregulated expression of miRNAs’ is observed and strictly linked to relevant human diseases (e.g. viral, immune, neurodegenerative and cardiovascular diseases, cancer). In this study we evaluated miRNAs’ expression modulation in corneal cells after treatment with different NGF bioformulations, by focusing the analysis on target genes and pathways as well. Tens of miRNAs were significantly dysregulated, mainly hypo-expressed, after treatment. Among those, 3 were shared by all the bioformulations, 37 by 2 of them, whereas 12, 16 and 20 were correlated to each single bioformulation. A total number of 91 unique pathways were identified by DIANA experimentally-supported Tarbase analysis: 46, including the neurotrophin signalling pathway, were shared by all three bioformulations, 28 by 2 of them, whereas 9, 2 and 6 were related to each single bioformulation. More than 2.000 experimentally supported target genes were identified. After focusing the analysis on the neurotrophin signalling pathway, no relevant differences were globally detected among the NGF bioformulations, all appearing to drive through proliferative and survival signals. However, one of the three bioformulations here used appeared to be more specific than the remaining two, as, when proportionally compared to them, it involved less significant miRNAs which, however, can target higher numbers of genes involved in this pathway. The same result was achieved when analysis was restricted to miRNAs’ sub-groups specifically related to each single bioformulation. Genemania gene and functional enrichment analysis further confirmed data. Futhermore, the gene list can be in depth analysed in order to identify targets suitable for further molecular analyses and validations, especially for each single bioformulation, with the aim to possibly identify target genes and functions specifically related to them. For this reason, more experiments by immunoblotting to evaluate expression levels of interesting target genes are ongoing. We examined the effects induced by different NGF bioformulations on epithelial corneal cells, by focusing the attention on miRNAs expression levels, and in silico target genes and pathways analysis. Although further functional studies to validate results are needed, original and novel insights about genes and epigenetics mechanisms induced by NGFs were provided. Our data may leave to hypothesize that NGF1 might be more specific and effective in inducing neurotrophin signalling pathway compared with NGF2 and NGF3. Importantly, given the putative role of miRNAs as biomarkers or therapeutic targets, this study makes available data potentially exploitable in clinical practice.
Studio dell'espressione di microRNA, geni target e pathways indotti da NGF in cellule epiteliali di cornea: confronto tra differenti bioformulazioni / Compagnoni, Chiara. - (2020 Jul 07).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11697/152310
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