18 research outputs found
Integrated microRNA and mRNA Signature Associated with the Transition from the Locally Confined to the Metastasized Clear Cell Renal Cell Carcinoma Exemplified by miR-146-5p
Background MicroRNAs (miRNAs) regulate gene expression by interfering
translation or stability of target transcripts. This interplay between miRNA
and their mRNA has been proposed as an important process in cancer development
and progression. We have investigated molecular networks impacted by predicted
mRNA targets of differentially expressed miRNAs in patients with clear cell
renal cell carcinoma (ccRCC) diagnosed with or without metastasis. Material
and Methods miRNA and mRNA microarray expression profiles derived from primary
ccRCC from patients with (16 samples) or without diagnosed metastasis (22
samples) were used to identify anti-correlated miRNA-mRNA interaction in
ccRCC. For this purpose, Ingenuity pathway analysis microRNA Target Filter,
which enables prioritization of experimentally validated and predicted mRNA
targets was used. By applying an expression pairing tool, the analysis was
focused on targets exhibiting altered expression in our analysis, finding
miRNAs and their target genes with opposite or same expression. The resulting
identified interactions were revalidated by RT-qPCR in another cohort of ccRCC
patients. A selection of the predicted miRNA-mRNA interactions was tested by
functional analyses using miRNA knockdown and overexpression experiments in
renal cancer cell lines. Results Among the significantly differentially
expressed miRNAs, we have identified three miRNAs (miR-146a-5p, miR-128a-3p,
and miR-17-5p) that were upregulated in primary tumors from patients without
metastasis and downregulated in primary tumors from patients with metastasis.
We have further identified mRNA targets, which expression were inversely
correlated to these 3 miRNAs, and have been previously experimentally
demonstrated in cancer setting in humans. Specifically, we showed that
CXCL8/IL8, UHRF1, MCM10, and CDKN3 were downregulated and targeted by miR-
146a-5p. The interaction between miR-146a-5p and their targets CXCL8 and UHRF1
was validated in cell culture experiments. Conclusions We identified novel
target genes of dysregulated miRNAs, which are involved in the transition from
primary RCC without metastases into tumors generating distant metastasis
Piwi-interacting RNAs as novel prognostic markers in clear cell renal cell carcinomas
Background Piwi-interacting RNAs (piRNAs) are small RNAs of 27–30 nucleotides
mapping to transposons or clustering in repeat genomic regions. Preliminary
studies suggest an important role in cancerogenesis. This study is the first
one investigating their prognostic impact in clear cell renal cell cancer
(ccRCC) patients. Methods Three piRNAs (piR-30924, piR-57125, and piR-38756)
selected on the basis of initial piRNA microarray analyses were determined
using RT-qPCR in non-metastatic (n = 76) and metastatic (n = 30) ccRCC tissue
at the time of nephrectomy in comparison to normal renal tissue (n = 77) and
tissue from distant ccRCC metastases (n = 13). Primary clinical end points
were recurrence-free and overall survival. Results piR-57125 showed lower
expression in metastatic than in non-metastatic tumors, whereas the expression
of piR-30924 and piR-38756 increased in metastatic tumors. The higher
expression of piR-30924 and piR-38756 as well as the lower expression of
piR-57125 in metastatic primary tumors were significantly associated with
tumor recurrence and overall survival. Multivariate Cox regression analyses
revealed both piR-30924 and piR-57125 as independent prognostic predictors.
This impact was even more pronounced in non-metastatic patients. Conclusions
This study demonstrates that the expression levels of these piRNAs in primary
non-metastatic and metastatic ccRCC tissue can serve as potential prognostic
biomarkers in combination with clinicopathological factors
Deregulated microRNAs and their diagnostic and prognostic impact in clear cell renal cell carcinoma
Hintergrund/Zielstellung: MicroRNAs (miRNAs) sind nicht-kodierende RNAs mit
einer Basenlänge von ca. 22 Nukleotiden. Es wird angenommen, dass sie 30 %
aller Gene regulieren und eine wichtige Rolle bei der Krebsentstehung und
-progression spielen. Der Wissensstand zur Bedeutung der miRNAs im
klarzelligen Nierenzellkarzinom war zu Beginn meiner Doktorarbeit sehr
begrenzt, sodass die Aufgabe darin bestand, deren mögliches diagnostisches und
prognostisches Potential fĂĽr das Nierenzellkarzinom zu ermitteln. Methodik:
Microarray-Analysen von Proben aus Karzinom- und umgebenden Normalgewebe, die
nach radikaler Nephrektomie gewonnen wurden, sowie von Knochenmetastasen-
Gewebe von metastasierten Nierenzellkarzinom-Patienten wurden in einer
exploratorischen Studie durchgefĂĽhrt. Die folgenden Validierungen erfolgten
mit der quantitativen reversen Transkriptions-Polymerase-Kettenreaktion (RT-
qPCR). Verschiedene spezielle Software (geNorm, NormFinder,
Prädiktionssuchmaschinen) und Statistikprogramme wurden für die Auswertung der
Daten genutzt. Ergebnisse: In der ersten Studie wurden die miR-28, miR-103 und
die miR-106a als die in den verschiedenen Gewebeproben am stabilsten
exprimierten miRNAs ermittelt. Die miRNA-Kombinationen aus der miR-28, miR-103
und miR-106a bzw. aus der miR-28 und miR-103 wurden als geeignete Referenz-
miRNAs-Kombinationen fĂĽr die relative Quantifizierung ermittelt. Bei
Gewebematerialmangel kann auch die miR-28 als EinzelmiRNA eingesetzt werden;
die oftmals genutzte RNU6B erwies sich als ungeeignetes Referenzgen. In der
zweiten Studie wurden, basierend auf den o.g. Normalisierungsansätzen, 30
deregulierte miRNAs zwischen den Tumor- und den Normalgewebeproben
identifiziert und mit der RT-qPCR validiert. Eine stufenweise verminderte
miRNA-Expression vom Normalgewebe über das primäre Tumorgewebe zum
Metastasengewebe war typisch. Nur sechs miRNAs zeigten eine erhöhte Expression
im Metastasengewebe im Vergleich zum Normalgewebe. Siebzehn miRNAs wurden als
neue miRNAs entdeckt, die mit der Metastasierung des Nierenzellkarzinoms
assoziiert sind und bisher in anderen Studien nicht beschrieben wurden.
Basierend auf diesen Daten und vorhergehenden Ergebnissen wurden jeweils im
Vergleich zum Normalgewebe vier stark ĂĽber- bzw. unterexprimierte miRNAs in
Karzinomgewebeproben von Nephrektomiepräparaten bei Patienten ohne (n=89) und
mit (n=22) Metastasen gemessen. Alle miRNAs erwiesen sich als geeignet,
malignes von nicht-malignem Gewebe zu differenzieren. Die beiden miRNAs
miR-122 und miR- 514 korrelierten signifikant mit dem Auftreten eines
Tumorrezidivs nach der Nephrektomie. Im Cox-Regressionsmodell mit klinisch-
pathologischen Standardvariablen erwies sich die miR-514 als eine signifikante
unabhängige Variable. Schlussfolgerungen: Basierend auf den vorliegenden
Studien kann geschlussfolgert werden, dass miRNA-Expressionsdaten nicht nur
eine wichtige Ergänzung zu den diagnostischen und prognostischen Informationen
durch die konventionellen klinischpathologischen Kenngrößen darstellen. Sie
ermöglichen außerdem neue, bisher verborgene Einsichten in molekulare Prozesse
der Krebsprogression und bieten damit neue Forschungsansätze auch in der
Therapie.Background/Objective: MicroRNAs (miRNAs) are non-protein coding RNAs of
approximately 22 nucleotides and are involved in the regulation of about 30%
of all genes. They play an important role in cancerogenesis and cancer
progression. Since their significance in clear cell renal cell carcinoma
(ccRCC) was limited at the beginning of my doctoral thesis, it was the aim to
evaluate the diagnostic and prognostic potential of miRNAs in ccRCC. Methods:
Microarray analyses of miRNAs from normal and cancerous samples of ccRCC
tissue collected after radical nephrectomy and from bone metastases of ccRCC
patients were performed to identify both invariant miRNAs as potential
referencemiRNAs for relative quantification and differentially expressed
miRNAs as diagnostic and prognostic indicators. The validation studies were
performed by quantitative reverse transcription polymerase chain reaction (RT-
qPCR) analyses. Different special software (geNorm, NormFinder, prediction
tools) and standard statistical programs were used for calculations. Results:
In the first study, miR-28, miR-103 and miR-106a were proved as the most
stably expressed miRNAs in the different tissue samples. Consequently, the
combinations of miR-28, miR-103, and miR-106a or miR-28 and miR-103 were
recommended as preferred normalizer approaches for relative quantification.
MiR-28 could be used as single normalizer in case of shortage of sample
material while RNU6B that is frequently used was unsuitable as normalizer. In
the second study, 30 miRNAs were identified to be particularly deregulated
between tumor and normal tissue samples. A stepwise down-regulation of miRNA
expression from normal over primary tumor to metastatic tissue was typical
while only six miRNAs were up-regulated in metastatic tissue in comparison to
normal tissue. Seventeen miRNAs were detected as novel miRNAs associated with
ccRCC metastasis that were not recognized as such in previous studies. Based
on these and previous findings, four up-regulated and four down-regulated
miRNAs in malignant and non-malignant samples after nephrectomy from patients
without (n=89) and with (n=22) metastases were measured. All miRNAs were found
to be suitable indicators to differentiate malignant from non-malignant
tissue. MiR-122 and miR-514 were significantly related to the recurrence risk
after nephrectomy and miR-514 was an independent prognostic variable in a
final Coxregression model together with clinicopathological variables.
Conclusions: Based on these studies, it could be shown that miRNA expression
data not only results in promising diagnostic and prognostic information in
completion to conventional clinicopathological data. They also provide novel
insights in yet unknown molecular processes of cancer progression and offer
new therapeutic strategies
Molecular tumor therapy: phase I/II network structure of the Society for Pediatric Oncology and Hematology in Germany
Background To enable the rapid and efficient implementation of early clinical trials for pediatric patients and provide comprehensive access to novel treatment options for pediatric relapse or high-risk patients, five regional phase I/II trial networks have been established under the auspices of the Society for Pediatric Oncology and Hematology (GPOH). The network structure secures, bundles and organizes required competencies and resources and has established processes to optimize operational trial efficiency. The INFORM study is a population-based molecular diagnostic study for children and adolescents with recurrent cancer in Germany and 11 other countries. Preliminary data show that precision medicine in pediatric oncology is possible in an international multicenter setting, provides clinical benefit for subgroups of patients and helps to identify hereditary predispositions, to refine diagnoses and to match patients in suitable phase I/II clinical trials. Conclusions The portfolio of innovative biomarker driven phase I/II clinical trials for children and adolescents with recurrent tumor diseases in Germany is currently limited. Therefore, the development of innovative, targeted therapy concepts and combination therapies is an important task in the coming years
Effect of miR-146a-5p on putative mRNA target levels in 786-O and ACHN cells.
<p>Application of miR-146a-5p mimic and miR-146a-5p inhibitor effects mRNA target expression in cell lines. Data were normalized with PPIA reference gene [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148746#pone.0148746.ref019" target="_blank">19</a>]. NC1 = negative control.</p
Effect of miR-146a-5p on CXCL8 protein levels in 786-O and ACHN cells.
<p>Application of miR-146a-5p mimic induced a significant reduction of CXCL8 protein expression in (A) 786-O cells and (B) ACHN cells, whereas a miR-146a-5p inhibitor rescued the CXCL8 expression levels in both cell lines. NC1 = negative control.</p
Interaction Network between 3 differentially expressed miRNAs (downregulated M1 vs M0, green) and differentially expressed mRNAs (upregulated M1 vs M0, red) from ccRCC patients.
<p>Interaction Network between 3 differentially expressed miRNAs (downregulated M1 vs M0, green) and differentially expressed mRNAs (upregulated M1 vs M0, red) from ccRCC patients.</p
Expression of putative mRNAs targeted of miR-146a-5p.
<p>The relative mRNA expression levels of the potential miR-146a-5p targets BRCA1, MCM10, CDKN3, CXCL8/IL8, and UHRF1 were measured in duplicates in a pool of normal renal tissue and tissue samples from primary ccRCC-M0 and ccRCC-M1 patients by RT-qPCR. Data were normalized with PPIA reference gene [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148746#pone.0148746.ref019" target="_blank">19</a>], (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148746#pone.0148746.s002" target="_blank">S2 Fig</a>). BRCA1, CDKN3, MCM10, CXCL8/IL8, and UHRF1 are lower in tissue samples of ccRCC without metastasis compared to ccRCC with metastasis.</p