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 für 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 durchgeführt. 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 für 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 über- 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