128 research outputs found
DNA methylation analysis of the evolution of Wilms tumour from its precursor nephrogenic rests
Recurrent loss of imprinting at 11p15, paucity of recurrent genetic mutations and associated nephrogenic rests (NR; precursor lesions that resemble embryonic kidney (EK)) implicate aberrant DNA methylation in tumourigenesis of paediatric Wilms tumour (WT) and predict that interrogation of the methylome, rather than the genome, is more likely to reveal tumour-specific biomarkers To test if aberrant DNA methylation is implicated in tumourigenesis, methylome analysis was performed on 36 normal kidney (NK), 22 NR, 36 WT and 4 EK, including 20 matched trios and 34 matched NK-WT pairs, using Illumina 450k arrays. Findings were validated with bisulfite-sequencing and RNA sequencing. This thesis describes the successful identification of changes in methylation that distinguish between tissue types. Through analysis of DNA methylation, NR formation was associated with gain of methylation at developmental loci related to Polycomb target binding sites. Evolution to WT was associated with increase in methylation variability in a subset of WTs (group-1), which also showed common changes in methylation in comparison to their associated NR, including silencing of novel tumour suppressor genes. Group-1 WTs were significantly enriched for bilateral cases whereas those in group-2 showed no differences in methylation compared to their associated NR. Comparison between NK and WT identified three DMRs of genome-wide significance (P<5x10-8) for use as tumour-specific biomarkers. As proof of principle for clinical utility, DMR-2 was successfully used in a case study to monitor tumour burden during treatment in cell-free serum DNA. This thesis concludes that methylation levels vary during WT evolution. As group-1 WT included all bilateral cases, our data suggests that methylation analysis could aid treatment planning in bilateral disease and that some WT may be candidates for epigenetic-modifier therapy. These findings define the first cell-free epigenetic biomarker for WT with potential for clinical utility
Integrated genomic characterization of pancreatic ductal adenocarcinoma
We performed integrated genomic, transcriptomic, and proteomic profiling of 150 pancreatic ductal adenocarcinoma (PDAC) specimens, including samples with characteristic low neoplastic cellularity. Deep whole-exome sequencing revealed recurrent somatic mutations in KRAS, TP53, CDKN2A, SMAD4, RNF43, ARID1A, TGFβR2, GNAS, RREB1, and PBRM1. KRAS wild-type tumors harbored alterations in other oncogenic drivers, including GNAS, BRAF, CTNNB1, and additional RAS pathway genes. A subset of tumors harbored multiple KRAS mutations, with some showing evidence of biallelic mutations. Protein profiling identified a favorable prognosis subset with low epithelial-mesenchymal transition and high MTOR pathway scores. Associations of non-coding RNAs with tumor-specific mRNA subtypes were also identified. Our integrated multi-platform analysis reveals a complex molecular landscape of PDAC and provides a roadmap for precision medicine
Identification of Differentially Expressed Genes in HPV AssociatedCancers Using Gene Expression, Tissue,and MicroRNA Microarrays
Infections with high-risk human papillomaviruses (HPVs) have been implicated in the pathogenesis of cervical carcinoma and a subset of squamous cell carcinoma of the head and neck (SCCHN). In this study, we compared the cellular gene expression profiles of HPV16-positive and HPV-negative oropharyngeal carcinomas with those of the normal oral epithelium. Using a high-density oligonucleotide microarray containing 22,215 human transcripts, we showed that 397 and 162 genes were differentially expressed in HPV16-positive and HPV-negative SCCHN, respectively, compared to the normal oral epithelium. Our studies also identified 59 differentially expressed genes in HPV16-positive SCCHN as compared to both HPV-negative SCCHN and normal oropharyngeal tissues. Such up-regulated genes included those involved in nuclear structure and meiosis (SYCP2), DNA repair (RFC5), and transcription regulation (ZNF238). Genes involved in proteolysis (KLK8) and signal transduction (CRABP2) were found to be down-regulated in HPV-positive SCCHN. Our results reveal specific gene expression patterns in HPV16-positive oropharyngeal squamous carcinomas and suggest that HPV infection could play an important etiologic role in these tumors. In another study using the same high-density microarray platform, we have analyzed the cellular gene expression profiles of five HPV-16 and two HPV-18 positive cervical cell lines, one HPV-negative cervical carcinoma cell line, and normal cervical tissue. Our results showed that 877 and 536 genes were differentially expressed in the HPV-positive cell lines compared to the normal cervix tissue and the HPV-negative cervical carcinoma cell line C-33A, respectively. We also found that a total of 57 genes were differentially expressed in the HPV-positive cell lines as compared to both the normal cervix and C-33A. Differentially expressed genes including those involved in cell proliferation such as the L-type amino acid transporter 1 (LAT1, also known as SLC7A5) and gene expression regulation like nucleosome assembly protein 1-like 3 (NAP1L3) were found to be affected for the first time in cervical cell lines. In situ hybridization of LAT1 and NAP1L3 mRNA performed using tissue-arrays (containing ~50 different cervical tumor samples per slide) showed that these genes are also affected in their expression in tumor tissues. These results could lead to the identification of new cellular pathways affected by the presence of HPV in cervical cells. We have also carried out studies to determine whether the expression of human microRNAs (miRNAs; small non-coding RNAs that have the ability to regulate gene expression) are affected by the presence of HPV DNA. For this purpose, we analyzed the expression of miRNAs in HPV-16 positive cervical cell lines and tissues. Twenty-seven miRNAs were differentially expressed in cervical cell lines containing integrated HPV-16 DNA compared to the normal cervix, while, only 6 miRNAs were differentially expressed in a cell line containing episomal HPV-16 DNA compared to the normal cervix. Furthermore, 10 miRNAs were affected in their expression in cell lines containing integrated HPV-16 DNA compared to C-33A. Interestingly, microRNA-218 (miR-218) was specifically underexpressed in cell lines, cervical lesions and cancer tissues containing integrated HPV-16 DNA as compared to both the HPV-negative cell line C-33A and the normal cervix. Expression of the HPV-16 E6 oncogene in transfected cells reduced miR-218 expression, and conversely, RNA interference of E6/E7 oncogenes in an HPV-16 positive cell line increased miR-218 expression. We also showed that miR-218 expression parallels that of the tumor suppressor gene SLIT2 whose intron encodes miR-218. Furthermore, exogenous expression of miR-218 in HPV-16 positive cell lines decreased expression of the epithelial-specific gene LAMB3 which is involved in cell migration and tumorigenicity. These findings demonstrate specific regulation of cellular miRNAs in the presence of an HPV oncogene and may contribute to a better understanding of molecular mechanisms involved in cervical carcinogenesis
Genomic and genetic dissection of thyroid cancer
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 17-06-2015Thyroid tumors can have two cellular origins and a variety of genetic drivers. Thus,
thyroid cancer (TC) is a complex and heterogeneous disease. As much of its etiology remains
poorly explored, TC represents an attractive model to study cancer disease process. Herein, we
have coupled exhaustive genomic dissection of an exceptional collection of human samples to
their genetic characterization, and comprehensive data analysis in order to address several
aspects of clinical interest.
In the first part of the study focused on follicular cell-derived cancer, by performing a
two-step association study involving 1,820 cases and 2,410 controls, we provide novel insights
into the genetic susceptibility of this disease. Apart from underscoring the importance of
9q22.33 locus in disease risk, we identify novel associations at 10q26.12 and 6q14.1 and
highlight that genetic heterogeneity between populations could be a part of this disease’s hidden
heritability. Moreover, we describe the genomic landscape of a total of 165 follicular cellderived
tumors including both papillary and follicular cases. We identify distinct molecular
subgroups closely related to oncogenic drivers, and explore the diagnostic and prognostic utility
of several genomic features. According to our results, the methylome and miRNome of benign
and malignant disease is largely overlapping, which prevents from diagnostic markers’
identification. Of note, elevated promoter methylation of WT1 and EI24, and aberrant
expression of let-7a and miR-192 could serve as potential novel molecular markers of shorter
time to progression.
The second part of this study is focused on the less frequent tumors of the gland arising
from C-cells, named medullary thyroid carcinomas (MTC). By characterizing the MTC
methylome, we have complemented the genomic dissection of an outstanding collection of 64
frozen and confirmed that this disease comprises of several molecular entities closely related to
the underlying mutations. Moreover, taking advantage of a series composed of 103 paraffin
embedded tumors we show that even tyrosine kinase inhibitors’ (TKI) targets expression differs
according to these mutations, and a priori genetic screening of MTC patients appears advisable
to guide the selection of the most suitable TKI treatment.
To summarize, we have performed genetic and genomic characterization of almost the
whole spectrum of thyroid tumors. Our studies have revealed much of molecular mechanisms
behind these tumors and shown that they tend to be tightly linked to the causal driver mutations.
On the whole, these results are yet another example of the great potential that lies in highthroughput
techniques to decipher disease etiology, and discover disease markers.Los tumores tiroideos pueden tener dos orígenes celulares y estar causados por una gran
variedad de mutaciones genéticas, siendo entonces el cáncer de tiroides (CT) una enfermedad
compleja y heterogénea. Gran parte de su etiología sigue siendo poco explorada, y el CT
representa un modelo atractivo de estudio. En este trabajo, se ha llevado a cabo una disección
exhaustiva de aspectos genómicos de una colección sobresaliente de muestras humanas
caracterizadas genéticamente, con el fin de abordar varios aspectos de interés clínico.
La primera parte del estudio se centra en el cáncer derivado de célula folicular.
Mediante un estudio de asociación en dos etapas que incluía 1,820 casos y 2,410 controles,
pudimos confirmar la importancia del locus 9q22.33 en el riesgo de la enfermedad, e
identificamos nuevas asociaciones en 10q26.12 y 6q14.1. Nuestros datos sugieren que la
heterogeneidad genética entre poblaciones podría en parte explicar la falta de replicación en
distintos estudios. Además, describimos las características genómicas de 165 tumores derivados
de célula folicular incluyendo tanto casos con patrón de crecimiento papilar como folicular.
Identificamos subgrupos moleculares específicamente relacionados con mutaciones concretas, y
exploramos la utilidad diagnóstica y pronostica de varias de las características genómicas
estudiadas. Así, el metiloma y el miRNoma de enfermedades benignas y malignas se solapan en
gran medida, lo cual impide la identificación de marcadores diagnósticos. Sin embargo, la
hipermetilación del promotor de WT1 y EI24, y la expresión aberrante de let-7a y miR-192
podrían servir como nuevos marcadores moleculares para predecir el tiempo hasta la progresión.
La segunda parte del estudio se centra en carcinomas tiroideos derivados de las células
C, llamados carcinoma medular de tiroides (CMT). Se caracterizó el metiloma de 48 CMT
congelados, complementando resultados previos de transcriptoma y miRNoma. Esta
enfermedad se compone de varias entidades moleculares estrechamente relacionadas con las
mutaciones subyacentes. Utilizando una colección de 103 tumores embebidos en parafina
demostramos que la expresión de dianas de inhibidores tirosina quinasas difiere de acuerdo a la
mutación. Por tanto, una caracterización genética previa de los pacientes con CMT parece
aconsejable para guiar la selección del tratamiento más adecuado.
En resumen, hemos caracterizado genética y genómicamente casi todo el espectro de
tumores tiroideos. Nuestros estudios han revelado mecanismos moleculares involucrados en su
desarrollo y han demostrado que los perfiles genómicos tienden a estar estrechamente
vinculados a mutaciones causales. En conjunto, estos resultados son una muestra del gran
potencial de las técnicas de alto rendimiento para descifrar la etiología de la enfermedad, y
descubrir marcadores de la enfermedad.This work was supported by the following grants and fellowships:
La Caixa/CNIO International PhD Fellowship, 2011-2015; Veronika Mančíková. Project PI11/01359 from Fondo de Investigaciones Sanitarias (FIS), Institute of Health.Carlos III.Project AP2775/2008 from Mutua Madrileña Foundation.
Project PI14/00240 from Fondo de Investigaciones Sanitarias (FIS), Institute of Health
Carlos III.
Project S2011/BMD-2328 from the Community of Madri
Next-generation sequencing identifies mechanisms of tumourigenesis caused by loss of SMARCB1 in Malignant Rhabdoid Tumours
PhD ThesisIntroduction: Malignant Rhabdoid Tumours (MRT) are unique malignancies caused by
biallelic inactivation of a single gene (SMARCB1). SMARCB1 encodes for a protein that
is part of the SWI/SNF chromatin remodelling complex, responsible for the regulation
of hundreds of downstream genes/pathways. Despite the simple biology of these
tumours, no studies have identified the critical pathways involved in tumourigenesis.
The understanding of downstream effects is essential to identifying therapeutic targets
that can improve the outcome of MRT patients.
Methods: RNA-seq and 450K-methylation analyses have been performed in MRT
human primary malignancies (n > 39) and in 4 MRT cell lines in which lentivirus was
used to re-express SMARCB1 (G401, A204, CHLA-266, and STA-WT1). The MRT cell
lines were treated with 5-aza-2 -deoxycytidine followed by global gene transcription
analysis (RNA-seq and 450K-methylation) to investigate how changes in methylation
lead to tumourigenesis.
Results: We show that primary Malignant Rhabdoid Tumours present a unique and
distinct expression/methylation profile which confirms that MRT broadly constitute a
single and different tumour type from other paediatric malignancies. However, despite
their common cause MRT can be can sub-group by location (i.e. CNS or kidney). We
observe that re-expression of SMARCB1 in MRT cell lines determines
activation/inactivation of specific downstream pathways such as IL-6/TGF beta. We
also observe a direct correlation between alterations in methylation and gene expression
in CD44, GLI2, GLI3, CDKN1A, CDKN2A and JARID after SMARB1 re-expression.
Loss of SMARCB1 also promotes expression of aberrant isoforms and novel transcripts
and causes genome-wide changes in SWI/SNF binding.
Conclusion: Next generation transcriptome and methylome analysis in primary MRT
and in functional models give us detailed downstream effects of SMARCB1 loss in
Malignant Rhabdoid Tumours. The integration of data from both primary and functional
models has provided, for the first time, a genome-wide catalogue of SMARCB1
tumourigenic changes (validated using systems biology). Here we show how a single
V
deletion of SMARCB1 is responsible for deregulation of expression, methylation status
and binding at the promoter regions of potent tumour-suppressor genes. The genes,
pathways and biological mechanisms indicated as key in tumour development may
ultimately be targetable therapeutically and will lead to better treatments for what is
currently one of the most lethal paediatric cancers.NECCR, Children with cancer UK, Brain Trust, Love Oliver,
CCLG, Karen and Iain Wark, The Smiley Ridley Fund, whose financial support made
this project possible
Unraveling the MYCN-driven transcriptional landscape of neuroblastoma : a cross-species integrated transcriptome approach
ince its identification in neuroblastoma, increasing evidence points towards a central role for MYCN in the biology of neuroblastoma tumors. As a consequence, targeting MYCN and its downstream functions would be a promising strategy to treat neuroblastoma patients. However, the development of inhibitors targeting MYC proteins has been challenging, as MYC/MYCN proteins are composed of two extended alpha-helices with no obvious surfaces for small molecule binding. Therefore, a thorough understanding of MYCN's functions and the upstream mechanisms regulating its expression would contribute to the development of compounds specifically targeting the impact of MYCN in neuroblastoma cells.
To address this issue, I initiated this thesis with the construction of an in vivo time-resolved miRNA and gene expression dataset of MYCN-driven neuroblastoma development, using the well-established TH-MYCN mouse model. After extensive validation of the dataset, I applied an exiting transcriptome-wide cross-species approach to search for transcriptional regulators that can be regarded as master regulators of neuroblastoma development. The corresponding miRNA expression dataset was exploited during the characterization of the complete MYCN- miRNA interactome in neuroblastoma using the powerful combination of this dataset and an unbiased 3'UTR reporter screen. I next characterized how MYCN affects the expression of LIN28B, a highly conserved RNA-binding protein, which is shown to induce MYCN expression in neuroblastoma via the suppression of the MYCN-targeting let-7 family of miRNAs.
Throughout this project, studying the genetic landscape of the well-established TH-MYCN mouse model has provided great insights in the perturbed miRNA-mRNA networks in neuroblastoma. Although representing an excellent and broadly used tool, this mouse model holds some limitations related to the transgene integration site, the anatomical location of the observed tumors, implementation of in vivo imaging and tumor incidence in different genetic backgrounds. I therefore present a novel mouse model with targeted Cre-conditional MYCN expression in the neural crest. Molecular characterization showed that the tumors arising in this model, strongly resemble the existing TH-MYCN mouse model and human neuroblastoma tumors. We expect that this new model, which addresses some of the major limitations of the TH-MYCN model, will play a major role in the investigation of neuroblastoma
Evaluation of blood-based microRNAs toward clinical use as biomarkers in common and rare diseases
According to the GLOBOCAN project of the International Agency for Research on Cancer, the top three common cancer diseases worldwide in the year 2020 were breast, lung and colorectal cancer. These are usually diagnosed via imaging methods (e.g. computer tomography) or invasive methods (e.g. biopsy). However, these techniques are potentially risky and expensive and thus not accessible to all patients, resulting in most cancers being detected in an advanced stage. Since the discovery of small non-coding RNAs and specifically microRNAs and their role as gene regulators, many researchers investigate their association with disease development. In particular, researchers examine body fluid based microRNAs which could present potential cost-effective and minimally- or non-invasive alternatives to the previously described established diagnosis methods.
This dissertation focuses on microRNAs and investigates their suitability as minimally-invasive blood-borne biomarkers for potential diagnostic purposes. More specifically, the goals of this work are (1) to implement a new method to predict novel microRNAs, (2) to understand stability and characteristics of these small non-coding RNAs, possibly relevant for the last goal, (3) to discover potential diagnostic biomarkers in common and rare diseases. The first goal was addressed by developing miRMaster, a web service to predict new microRNAs. The tool uses machine learning and high-throughput sequencing data to find microRNA candidates that follow the known biogenesis pathways. The second goal was pursued in four publications. First, we performed a large scale evaluation of miRMaster by generating a high-resolution map of the human small non-coding RNA transcriptome for which we analyzed and validated potential microRNA candidates. Next, we examined the influence of seasonal effects on microRNA expression profiles and observed the largest difference between spring and the other seasons. Additionally, we evaluated the evolutionary conservation of small non-coding RNAs in zoo animals and showed that the distribution of sncRNA classes varies across species, while common microRNA families are present in more diverse organisms than assumed so far. Furthermore, we analyzed if microRNAs are technically stable, and whether biological variation is preserved when using capillary dried blood spots as an alternative sample collection device to venous blood specimens. Finally, we investigated the suitability of microRNAs as biomarkers for two diseases: lung cancer and Marfan disease. We identified blood-borne biomarker candidates for lung cancer detection in a large-scale multi-center study via machine learning. For the rare Marfan disease we analyzed the paired messenger RNA and microRNA expression levels in whole-blood samples. This highlighted several significantly deregulated microRNAs and messenger RNAs, which we subsequently validated in an independent cohort.
In summary, this thesis provides valuable results toward potential clinical use of microRNAs, and the herein described projects represent comprehensive analyses of them from different perspectives: starting with microRNA discovery, addressing various technical and biological questions and ending with the potential use as biomarkers.Nach Angaben des GLOBOCAN-Projekts der International Agency
for Research on Cancer sind die drei häufigsten Krebserkrankungen
weltweit im Jahr 2020 Brust-, Lungen- und Darmkrebs. Diese werden in
der Regel durch bildgebende Verfahren (z.B. Computertomographie)
oder invasive Methoden (z.B. Biopsie) diagnostiziert. Diese Verfahren
sind jedoch potenziell risikoreich und teuer und daher nicht für alle
Patienten zugänglich. Dies führt dazu, dass die meisten Krebsarten
erst in einem fortgeschrittenen Stadium entdeckt werden. Seit der
Entdeckung der kurzen nichtkodierenden RNAs und insbesondere
der microRNAs und ihrer Rolle als Genregulatoren untersuchen viele
Forscher ihren Zusammenhang mit der Krankheitsentwicklung. Insbesondere
untersuchen die Forscher die in Körperflüssigkeiten vorkommenden
microRNAs, die potenziell kosteneffiziente und minimal- oder
nicht-invasive Alternativen zu den bisher beschriebenen etablierten
Diagnosemethoden darstellen könnten.
Diese Dissertation konzentriert sich auf microRNAs und untersucht
deren Eignung als minimal-invasive blutbasierte Biomarker
für potenzielle diagnostische Zwecke. Genauer gesagt sind die Ziele
dieser Arbeit (1) die Implementierung einer neuen Methode zur
Vorhersage neuartiger microRNAs, (2) das Verständnis über die Stabilität
und Charakteristika dieser kurzen nicht-kodierenden RNAs, die
möglicherweise für das nächste Ziel relevant sind, (3) die Entdeckung
potenzieller diagnostischer Biomarker für verschiedene Anwendungen.
Das erste Ziel wurde durch die Entwicklung von miRMaster verfolgt,
einem Webdienst zur Vorhersage neuer microRNAs. Das Tool nutzt
maschinelles Lernen und Hochdurchsatz-Sequenzierungsdaten, um
microRNA-Kandidaten zu finden, die den bekannten Wege der
Biogenese folgen. Das zweite Ziel wurde in vier Veröffentlichungen
verfolgt. Zunächst führten wir eine groß angelegte Evaluierung
von miRMaster durch, indem wir eine High-Resolution Map des
menschlichen Transkriptoms kurzer nichtkodierender RNAs erstellten,
für die wir potenzielle microRNA-Kandidaten analysierten und
validierten. Anschließend untersuchten wir den Einfluss saisonaler
Effekte auf die microRNA-Expressionsprofile und beobachteten
den größten Unterschied zwischen dem Frühling und den anderen
Jahreszeiten. Darüber hinaus untersuchten wir die evolutionäre
Erhaltung kurzer nichtkodierender RNAs in Zoo-Tieren und zeigten,
dass die Verteilung der kurzer nichtkodierenden RNA-Klassen zwischen
den Arten variiert, während gemeinsame microRNA-Familien
in verschiedeneren Organismen vorkommen als bisher angenommen.
Darüber hinaus analysierten wir, ob microRNAs technisch
stabil sind und ob die biologische Variation erhalten bleibt, wenn
kapillares Trockenblut als alternatives Probenentnahmeverfahren zu
venösen Blutproben verwendet werden. Schließlich untersuchten wir
die Eignung von microRNAs als Biomarker für zwei Krankheiten:
Lungenkrebs und Marfan-Krankheit. In einer groß angelegten multizentrischen
Studie identifizierten wir mit Hilfe von maschinellem
Lernen Biomarker-Kandidaten aus dem Blut für die Erkennung von
Lungenkrebs. Für die seltene Marfan-Krankheit analysierten wir die
gepaarten Expressionsniveaus von messengerRNA und microRNA
in Vollblutproben. Dabei wurden mehrere signifikant deregulierte
microRNAs und messengerRNAs festgestellt, die wir anschließend in
einer unabhängigen Kohorte validierten.
Zusammenfassend lässt sich sagen, dass diese Arbeit wertvolle
Ergebnisse im Hinblick auf die potenzielle klinische Verwendung von
microRNAs liefert. Die hier beschriebenen Projekte stellen umfassende
Analysen aus verschiedenen Blickwinkeln dar: angefangen bei der
Entdeckung von microRNAs, über verschiedene technische und biologische
Fragen bis hin zur potenziellen Verwendung als Biomarker
MicroRNA regulation of podocyte insulin sensitivity
Diabetic Nephropathy (DN) is a devastating complication of diabetes, and is the leading cause of end stage renal failure in the UK. Uncovering mechanistic pathways in DN pathogenesis is vital in establishing new therapeutic targets to prevent its progression.
Podocyte-specific insulin resistance in mice leads to a renal injury that mimics that seen in diabetic kidney disease, indicating that podocyte insulin signalling may be of critical importance in the development of DN. MicroRNAs (miRNAs) are post-transcriptional gene regulators that demonstrate aberrant expression in multiple diabetic models, and are implicated in the development of insulin resistance in liver, fat and muscle. The aim of this work was to establish the role of miRNAs in the regulation of podocyte insulin signalling.
This thesis details the differential microRNA expression of an in vitro model of podocyte insulin resistance, and the subsequent validation of miR-155-5p as an important regulator of podocyte insulin sensitivity. MiR-155 was upregulated in insulin-resistant podocytes, and in the urine of patients with DN. Furthermore, overexpression of miR-155 in podocytes resulted in reduction in PI3K/Akt signalling and abrogation of glucose uptake in vitro. Bioinformatic analyses were used to identify putative miR-155 targets. PIK3R1 and CSF1R were confirmed to demonstrate miR-155 induced repression, hypothesised to result in podocyte insulin resistance via negative regulation of PI3K signalling.
Whole glomerular miRNA sequencing from the db/db mouse indicated that changes associated with established diabetic pathways of oxidative stress, inflammation and fibrosis are transcriptionally activated as early as 4 weeks. These findings support the hypothesis that changes in miRNA expression are an initiating insult in DN, and highlight miRNAs as potential therapeutic targets to arrest disease development
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