Tesi etd-03302022-190134
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Tipo di tesi
Dottorato
Autore
DATTILO, ALESSIA
URN
etd-03302022-190134
Titolo
miRs-320 family and miR-196a-5p: novel circulating biomarkers of lipodystrophy
Settore scientifico disciplinare
BIO/11
Corso di studi
Istituto di Scienze della Vita - TRANSLATIONAL MEDICINE 2018
Commissione
relatore Prof. RECCHIA, FABIO ANASTASIO
Parole chiave
- Nessuna parola chiave trovata
Data inizio appello
17/10/2022;
Disponibilità
parziale
Riassunto analitico
Lipodystrophy (LD) indicates a group of rare disorders, with generalized or partial loss of white adipose tissue (WAT) often associated with metabolic derangements. Heterogeneity/wide spectrum of the disease and lack of biomarkers make diagnosis often difficult. MicroRNAs are important to maintain a correct WAT function and WAT is a source of circulating miRNAs (cmiRs). Aim of this study was to investigate if LD can result in altered abundance of specific cmiRs.
To this end, a discovery step of the study was performed in two small cohorts of subjects, LD versus healthy controls (HC), using NGS-based cmiRs profiling. Validation step conducted in larger cohorts of LD and HC, employing quantitative PCR (qPCR), showed a differential expression between LD and HC of cmiRs from 320 family and miR-196a-5p, with miR-320a-3p, -320-b, -320-c, -320-e being upregulated, while -320d and miR-196a-5p downregulated. The power as classifiers of both miRNAs from 320 family and miR-196a-5p was more marked in the most extreme (Congenital Generalized LD, CGL) and well-defined (Dunningan, FPLD2) forms of LD; on the other exteme, for FPLD1 (or Kobberling Syndrome), characterized by subjects with a very wide spectrum of the disorder, our miRNAs are useless as classifiers.
Subsequent studies were conducted to search for a more in depth characterization of the clinical and biological significance of our findings. Eventhough a similar experimental design was applied to both classes of miRNAs, we herein selected the most significant results, which for miRs 320 privilege the relationship with the clinical and gene ontology aspects, wherase for miR-196a-5p disclose interesting biological insights.
miRs-320
We found that cmiRs-320 family show significant inverse relationships with plasma leptin and BMI, typically low in LD and the hepatic enzymes and the marker of inflammation CRP are inversely related to cmiR-320d. miR320a-3p shows the highest number of significant associations with clinical parameters.
Gene ontology analysis revealed that miRs-320 target proteins are highly aggregated in defining the capacity of cells to build contacts and strengthen the architecture of tissues.
In conclusion, cmiRs 320 constitute novel biomarkers of LD, able to distinguish affected versus non affected for most of its subtypes. High circulating levels of miR-320a-3p, -320-b, -320-c, -320-e and
low levels of cmiR-320d predicts the salient aspects of the disease, i.e. reduced WAT mass and function, and further low levels of cmiR-320d are associated to altered hepatic profile and higher inflammation, two typical derangements found in LD. In silico analysis suggest extracellular matrix components as targets of miRs-320 family and cell-cell adhesion as a process regulated by 320 miRNAs targets, disclosing a novel route to investigate the origin of WAT loss.
miR-196a-5p
Downregulation of miR-196a-5p was revealed by qPCR in the residual subcutaneous WAT of LD compared to HC subjects.
During adipogenesis of SGBS (Simpson-Golabi-Behmel syndrome) cells, miR-196a-5p expression is characterized by high expression in the early stages of differentiation and a significant reduction in the mid-final stages of the process. When SGBS cells are treated with protease inhibitors (PI), which blunt their adipogenic capacity, thus generating a model of adipocyte dysfunction, the reduction of miR-196a-5p is anticipated. We interfered with this anticipated downregulation by transfecting undifferentiated SGBS cells with a miR-196a-5p mimic, and we evaluated the expression of typical markers of adipocyte terminal differentiation upon completion of the adipogenic process. Preliminary results indicate that overexpression of miR-196a-5p in PI treated SGBS cells results in higher adiponectin, leptin, fatty acid synthase and perilipin, compared to PI untransfected cells.
In conclusion, downregulation of cmiR-196a-5p constitutes a novel biomarker of LD and of most of its subtypes. The reduction found in the WAT of LD subjects suggests that the adipose organ contributes to its altered circulating levels. miR-196a-5p reduced expression is associated to WAT dysfunction as suggested by data obtained in vivo and in vitro. Interfering with the modulation of this miRNA may constitute a novel route to repristinate a correct adipocyte function and further experiments are needed to explore the therapeutic potential of its action.
To this end, a discovery step of the study was performed in two small cohorts of subjects, LD versus healthy controls (HC), using NGS-based cmiRs profiling. Validation step conducted in larger cohorts of LD and HC, employing quantitative PCR (qPCR), showed a differential expression between LD and HC of cmiRs from 320 family and miR-196a-5p, with miR-320a-3p, -320-b, -320-c, -320-e being upregulated, while -320d and miR-196a-5p downregulated. The power as classifiers of both miRNAs from 320 family and miR-196a-5p was more marked in the most extreme (Congenital Generalized LD, CGL) and well-defined (Dunningan, FPLD2) forms of LD; on the other exteme, for FPLD1 (or Kobberling Syndrome), characterized by subjects with a very wide spectrum of the disorder, our miRNAs are useless as classifiers.
Subsequent studies were conducted to search for a more in depth characterization of the clinical and biological significance of our findings. Eventhough a similar experimental design was applied to both classes of miRNAs, we herein selected the most significant results, which for miRs 320 privilege the relationship with the clinical and gene ontology aspects, wherase for miR-196a-5p disclose interesting biological insights.
miRs-320
We found that cmiRs-320 family show significant inverse relationships with plasma leptin and BMI, typically low in LD and the hepatic enzymes and the marker of inflammation CRP are inversely related to cmiR-320d. miR320a-3p shows the highest number of significant associations with clinical parameters.
Gene ontology analysis revealed that miRs-320 target proteins are highly aggregated in defining the capacity of cells to build contacts and strengthen the architecture of tissues.
In conclusion, cmiRs 320 constitute novel biomarkers of LD, able to distinguish affected versus non affected for most of its subtypes. High circulating levels of miR-320a-3p, -320-b, -320-c, -320-e and
low levels of cmiR-320d predicts the salient aspects of the disease, i.e. reduced WAT mass and function, and further low levels of cmiR-320d are associated to altered hepatic profile and higher inflammation, two typical derangements found in LD. In silico analysis suggest extracellular matrix components as targets of miRs-320 family and cell-cell adhesion as a process regulated by 320 miRNAs targets, disclosing a novel route to investigate the origin of WAT loss.
miR-196a-5p
Downregulation of miR-196a-5p was revealed by qPCR in the residual subcutaneous WAT of LD compared to HC subjects.
During adipogenesis of SGBS (Simpson-Golabi-Behmel syndrome) cells, miR-196a-5p expression is characterized by high expression in the early stages of differentiation and a significant reduction in the mid-final stages of the process. When SGBS cells are treated with protease inhibitors (PI), which blunt their adipogenic capacity, thus generating a model of adipocyte dysfunction, the reduction of miR-196a-5p is anticipated. We interfered with this anticipated downregulation by transfecting undifferentiated SGBS cells with a miR-196a-5p mimic, and we evaluated the expression of typical markers of adipocyte terminal differentiation upon completion of the adipogenic process. Preliminary results indicate that overexpression of miR-196a-5p in PI treated SGBS cells results in higher adiponectin, leptin, fatty acid synthase and perilipin, compared to PI untransfected cells.
In conclusion, downregulation of cmiR-196a-5p constitutes a novel biomarker of LD and of most of its subtypes. The reduction found in the WAT of LD subjects suggests that the adipose organ contributes to its altered circulating levels. miR-196a-5p reduced expression is associated to WAT dysfunction as suggested by data obtained in vivo and in vitro. Interfering with the modulation of this miRNA may constitute a novel route to repristinate a correct adipocyte function and further experiments are needed to explore the therapeutic potential of its action.
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