Epithelial-mesenchymal transition-associated microRNA/mRNA signature is linked to metastasis and prognosis in clear-cell renal cell carcinoma

Clear-cell renal cell carcinomas (ccRCCs) are genetically heterogeneous tumors presenting diverse clinical courses. Epithelial-mesenchymal transition (EMT) is a crucial process involved in initiation of metastatic cascade. The aim of our study was to identify an integrated miRNA/mRNA signature associated with metastasis and prognosis in ccRCC through targeted approach based on analysis of miRNAs/mRNAs associated with EMT. A cohort of 230 ccRCC was included in our study and further divided into discovery, training and validation cohorts. EMT markers were evaluated in ccRCC tumor samples, which were grouped accordingly to EMT status. By use of large-scale miRNA/mRNA expression profiling, we identified miRNA/mRNA with significantly different expression in EMT-positive tumors and selected 41 miRNAs/mRNAs for training phase of the study to evaluate their diagnostic and prognostic potential. Fifteen miRNAs/mRNAs were analyzed in the validation phase, where all evaluated miRNA/mRNA candidates were confirmed to be significantly deregulated in tumor tissue. Some of them significantly differed in metastatic tumors, correlated with clinical stage, with Fuhrman grade and with overall survival. Further, we established an EMT- based stage-independent prognostic scoring system enabling identification of ccRCC patients at high-risk of cancer-related death. Finally, we confirmed involvement of miR-429 in EMT regulation in RCC cells in vitro

Supplementary material for the article: Macháčková, K.; Chrudinová, M.; Radosavljević, J.; Potalitsyn, P.; Křížková, K.; Fábry, M.; Selicharová, I.; Collinsová, M.; Brzozowski, A. M.; Žáková, L.; et al. Converting Insulin-like Growth Factors 1 and 2 into High-Affinity Ligands for Insulin Receptor Isoform A by the Introduction of an Evolutionarily Divergent Mutation. Biochemistry 2018, 57 (16), 2373– 2382. https://doi.org/10.1021/acs.biochem.7b01260

Supplementary material for: [https://doi.org/10.1021/acs.biochem.7b01260]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2135

Changes in Hematologic and Coagulation Profiles in Rabbits with Right-ventricle Pacing.

Abstract Objectives: The aim of this study was to evaluate changes in hematology and coagulation in rabbits with right-ventricle pacing without medication. Animals and methods: Blood was collected from ten non-anesthetized male rabbits from the jugular vein before and one month after pacemaker placement. Total erythrocyte, leukocyte and platelet count, hemoglobin, hematocrit and differential leukocyte count were done on automatic veterinary flow cytometry hematologic analyzer. Prothrombin time, activated partial thromboplastin time, fibrinogen level, D-dimers and kaolin-activated thromboelastography was measured from citrated blood. Results: We found an increase in red blood cell mass and decrease in platelet count, while coagulation tests did not diff er between samplings. Conclusion: Right-ventricle pacing seems to have no influence on hemostasis in rabbits

Changes in Hematologic and Coagulation Profiles in Rabbits with Right-ventricle Pacing

The aim of this study was to evaluate changes in hematology and coagulation in rabbits with right-ventricle pacing without medication

MicroRNA Biogenesis Pathway Genes Are Deregulated in Colorectal Cancer

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Each step of their production and maturation has to be strictly regulated, as any disruption of control mechanisms may lead to cancer. Thus, we have measured the expression of 19 genes involved in miRNAs biogenesis pathway in tumor tissues of 239 colorectal cancer (CRC) patients, 17 CRC patients with liver metastases and 239 adjacent tissues using real-time PCR. Subsequently, the expression of analyzed genes was correlated with the clinical-pathological features as well as with the survival of patients. In total, significant over-expression of all analyzed genes was observed in tumor tissues as well as in liver metastases except for LIN28A/B. Furthermore, it was shown that the deregulated levels of some of the analyzed genes significantly correlate with tumor stage, grade, location, size and lymph node positivity. Finally, high levels of DROSHA and TARBP2 were associated with shorter disease-free survival, while the over-expression of XPO5, TNRC6A and DDX17 was detected in tissues of patients with shorter overall survival and poor prognosis. Our data indicate that changed levels of miRNA biogenesis genes may contribute to origin as well as progression of CRC; thus, these molecules could serve as potential therapeutic targets

Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes

<div><p>Background</p><p>Carriers of mutations in hereditary cancer predisposition genes represent a small but clinically important subgroup of oncology patients. The identification of causal germline mutations determines follow-up management, treatment options and genetic counselling in patients’ families. Targeted next-generation sequencing-based analyses using cancer-specific panels in high-risk individuals have been rapidly adopted by diagnostic laboratories. While the use of diagnosis-specific panels is straightforward in typical cases, individuals with unusual phenotypes from families with overlapping criteria require multiple panel testing. Moreover, narrow gene panels are limited by our currently incomplete knowledge about possible genetic dispositions.</p><p>Methods</p><p>We have designed a multi-gene panel called CZECANCA (CZEch CAncer paNel for Clinical Application) for a sequencing analysis of 219 cancer-susceptibility and candidate predisposition genes associated with frequent hereditary cancers.</p><p>Results</p><p>The bioanalytical and bioinformatics pipeline was validated on a set of internal and commercially available DNA controls showing high coverage uniformity, sensitivity, specificity and accuracy. The panel demonstrates a reliable detection of both single nucleotide and copy number variants. Inter-laboratory, intra- and inter-run replicates confirmed the robustness of our approach.</p><p>Conclusion</p><p>The objective of CZECANCA is a nationwide consolidation of cancer-predisposition genetic testing across various clinical indications with savings in costs, human labor and turnaround time. Moreover, the unified diagnostics will enable the integration and analysis of genotypes with associated phenotypes in a national database improving the clinical interpretation of variants.</p></div

Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes - Fig 7

<p><b>Comparison of variant detection (shown as values of variant allelic fraction; AF) in DNA reference standards</b> (NA12878, NA24149, NA24385, NA24631 and NA24143) obtained from CZECANCA analysis (x-axis) and AF from VCF files for these standards downloaded from <a href="http://jimb.stanford.edu/giab/" target="_blank">http://jimb.stanford.edu/giab/</a> (y-axis). The graph shows all variants with GATK quality >100 reached in CZECANCA analysis (including FP variants) and undetected (FN) variants. Heterozygote variants clustered in the center, while homozygote variants in right upper corner. Variant distribution was partially influenced by the differences in mean sequencing coverage targeting 100X and 300X in CZECANCA and DNA reference standards VCFs, respectively. The number of TP, TN, FP, FN, and total number of variant (= CZECANCA target) was used to calculate of sensitivity, specificity, and accuracy of CZECANCA analysis.</p