Die Rolle der Cyclin-abhängigen Kinase 18 (CDK18) im klarzelligen Nierenzellkarzinom

The most common and one of the more aggressive types of kidney cancer is clear cell renal cell carcinoma. It is characterized by sporadic occurrence, poor prognosis, and high resistance to therapies, which necessitates the discovery of new biomarkers for improving diagnostics and prognostics. The aim of the study was to identify a panel of genes whose mRNA is strongly upregulated in clear cell carcinoma tissue, in order to develop a qPCR detection assay based on their potential differential expression in the blood of cancer patients compared to healthy individuals. A further aim was to functionally characterize a novel gene in cell lines representing this cancer. The construction of the gene panel was performed by a bioinformatic analysis of several databases containing tissue (tumor and normal) and blood expression (from healthy individuals) of all genes in the genome. The presence of selected genes was tested in tissue and blood of patients and healthy individuals by RT-qPCR. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) /Cas9 system enabled the generation of stable knockout clones for a loss-of-function analysis, and RNA sequencing allowed for the global transcriptome analysis of the knockout condition, revealing the possible mechanism of action of the investigated gene. A ranked list of genes overexpressed in clear cell carcinoma tissue compared to adjacent normal kidney tissue was produced, among them CDK18 (cyclin-dependent kinase 18), CCND1 and LOX. Two genes, CDK18 and CCND1 were underexpressed in the blood of clear cell carcinoma patients, and LOX showed a tendency towards upregulation in metastatic compared to non-metastatic blood samples. CDK18 knockout in two renal cancer cell lines led to a reduced proliferation rate, possibly via effects on WDR77 and SOAT1, the former being downregulated, and the second showing a tendency towards downregulation in the knockout condition.Die häufigste und eine der aggressiveren Formen von Nierentumoren ist das klarzellige Nierenzellkarzinom. Charakteristika sind sporadisches Auftreten, schlechte Prognose und hohe Therapieresistenz, und deswegen ist die Entdeckung neuer Biomarker zur Verbesserung von Diagnostik und Prognose erforderlich. Das Ziel der Studie war, eine Gruppe von Genen zu identifizieren, deren mRNA im klarzelligen Nierenzellkarzinom stark hochreguliert ist, um einen qPCR-Assay zu entwickeln, der auf ihrer potenziellen differenziellen Expression im Blut von Krebspatienten im Vergleich zu gesunden Personen basiert. Ferner sollte, ein neues Gen in Zelllinien, die diesen Tumor repräsentieren, funktionell charakterisiert werden. Methoden: Die Gruppe von Genen wurde durch bioinformatische Analyse mehrerer Datenbanken, die die Gewebe- und Blutexpression aller Gene enthalten, herausgefiltert. Die Expression von ausgewählten Genen wurde in Gewebe und Blut von Patienten und gesunden Personen durch RT-qPCR bestimmt. Das CRISPR/Cas9-System ermöglichte die Erzeugung von stabilen Knockout-Klonen für die Funktionsverlustanalyse, und die RNA-Sequenzierung ermöglichte die globale Transkriptomanalyse des Knockout-Zustands und die Aufdeckung des möglichen Wirkmechanismus des untersuchten Gens. Ergebnisse: Eine Rangliste von Genen, die in klarzelligem Nierenzellkarzinomgewebe im Vergleich zu benachbartem normalem Nierengewebe überexprimiert sind, wurde erstellt, darunter CDK18, CCND1 und LOX. Zwei Gene, CDK18 und CCND1, waren im Blut von Klarzellkarzinompatienten vermindert exprimiert, und LOX zeigt eine Tendenz zur Hochregulation bei metastatischen im Vergleich zu nicht-metastatischen Blutproben. CDK18 Knockout in zwei Nierenkrebszelllinien führte zu einer reduzierten Proliferationsrate, möglicherweise durch Effekte auf WDR77 und SOAT1, wobei das erste herunterreguliert war und das zweite eine Tendenz zur Herunterregulierung im Knockout-Zustand zeigte. Schlussfolgerungen: Diese Studie veranschaulicht die Schwierigkeit, tumorspezifische mRNAs im Blut nachzuweisen, und zeigte paradoxerweise eine verminderte Expression von zwei Genen im Blut von Klarzellkarzinompatienten entgegen der Überexpression im Gewebe. Die Studie konnte den Einfluss von CDK18 auf die Tumorzellproliferation belegen und einen möglichen Mechanismus dafür aufzeigen, der noch näher erforscht werden sollte

Limited utility of qPCR-based detection of tumor-specific circulating mRNAs in whole blood from clear cell renal cell carcinoma patients

BACKGROUND: RNA sequencing data is providing abundant information about the levels of dysregulation of genes in various tumors. These data, as well as data based on older microarray technologies have enabled the identification of many genes which are upregulated in clear cell renal cell carcinoma (ccRCC) compared to matched normal tissue. Here we use RNA sequencing data in order to construct a panel of highly overexpressed genes in ccRCC so as to evaluate their RNA levels in whole blood and determine any diagnostic potential of these levels for renal cell carcinoma patients. METHODS: A bioinformatics analysis with Python was performed using TCGA, GEO and other databases to identify genes which are upregulated in ccRCC while being absent in the blood of healthy individuals. Quantitative Real Time PCR (RT-qPCR) was subsequently used to measure the levels of candidate genes in whole blood (PAX gene) of 16 ccRCC patients versus 11 healthy individuals. PCR results were processed in qBase and GraphPadPrism and statistics was done with Mann-Whitney U test. RESULTS: While most analyzed genes were either undetectable or did not show any dysregulated expression, two genes, CDK18 and CCND1, were paradoxically downregulated in the blood of ccRCC patients compared to healthy controls. Furthermore, LOX showed a tendency towards upregulation in metastatic ccRCC samples compared to non-metastatic. CONCLUSIONS: This analysis illustrates the difficulty of detecting tumor regulated genes in blood and the possible influence of interference from expression in blood cells even for genes conditionally absent in normal blood. Testing in plasma samples indicated that tumor specific mRNAs were not detectable. While CDK18, CCND1 and LOX mRNAs might carry biomarker potential, this would require validation in an independent, larger patient cohort

The SIB Swiss Institute of Bioinformatics' resources: focus on curated databases

The SIB Swiss Institute of Bioinformatics (www.isb-sib.ch) provides world-class bioinformatics databases, software tools, services and training to the international life science community in academia and industry. These solutions allow life scientists to turn the exponentially growing amount of data into knowledge. Here, we provide an overview of SIB's resources and competence areas, with a strong focus on curated databases and SIB's most popular and widely used resources. In particular, SIB's Bioinformatics resource portal ExPASy features over 150 resources, including UniProtKB/Swiss-Prot, ENZYME, PROSITE, neXtProt, STRING, UniCarbKB, SugarBindDB, SwissRegulon, EPD, arrayMap, Bgee, SWISS-MODEL Repository, OMA, OrthoDB and other databases, which are briefly described in this article