[Usage of Cerebrospinal Fluid for microRNA Analysis]. / Vyuzití mozkomísního moku pro analýzu mikroRNA.

BACKGROUNDS: Deregulated levels of miRNAs, short noncoding RNAs associated with pathogenesis of many diseases, have been observed in cerebrospinal fluid (CSF). Therefore, the analysis of CSF miRNAs in patients affected by tumors of central nervous system (CNS) might help to develop new diagnostic platform enabling more precise diagnosis. Thus, in our study we tried to optimize methodical approaches to be used for miRNA detection as RNA isolation and selection of suitable technology for global high-throughput miRNA profiling. MATERIAL AND METHODS: In the optimization phase of RNA isolation from CSF, various commercially available kits with different protocol modifications were compared. Two quantitative polymerase chain reaction panels and Next Generation Sequencing method were tested for selection of the most suitable method for miRNA comprehensive profiling. RESULTS: The Urine miRNA Purification kit (Norgen) and Next Generation Sequencing was selected as the most suitable kit for RNA extraction from CSF and method for miRNA comprehensive profiling, resp. CONCLUSION: We established a protocol for RNA isolation and miRNA comprehensive profiling in CSF clinical specimens.Key words: brain neoplasm - cerebrospinal fluid - microRNA The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. This study was supported by Ministry of Health of the Czech Republic, grant No. 15-34553A. All rights reserved.Submitted: 19. 3. 2018Accepted: 10. 4. 2018.

The biobanking research infrastructure BBMRI_CZ: a critical tool to enhance translational cancer research.

We introduce the national research biobanking infrastructure, BBMRI_CZ. The infrastructure has been founded by the Ministry of Education and became a partner of the European biobanking infrastructure BBMRI.eu. It is designed as a network of individual biobanks where each biobank stores samples obtained from associated healthcare providers. The biobanks comprise long term storage (various types of tissues classified by diagnosis, serum at surgery, genomic DNA and RNA) and short term storage (longitudinally sampled patient sera). We discuss the operation workflow of the infrastructure that needs to be the distributed system: transfer of the samples to the biobank needs to be accompanied by extraction of data from the hospital information systems and this data must be stored in a central index serving mainly for sample lookup. Since BBMRI_CZ is designed solely for research purposes, the data is anonymised prior to their integration into the central BBMRI_CZ index. The index is then available for registered researchers to seek for samples of interest and to request the samples from biobank managers. The paper provides an overview of the structure of data stored in the index. We also discuss monitoring system for the biobanks, incorporated to ensure quality of the stored samples.

Significant overexpression of Hsp110 gene during colorectal cancer progression.

Colorectal cancer (CRC) is one of the most frequent malignant diseases in the world. Metastatic spread of the cancer to the lymph nodes is a crucial factor for progression and therapeutic management of the disease. We analysed gene expression profiles of CRC patiens by low-density cancer-focused oligonucleotide microarrays to identify new predictive markers of the extent of the disease and for better understanding of CRC progression. Relative expression levels of 440 genes known to be involved in cancer biology were obtained by low-density oligonucleotide microarrays from 20 tumor samples. Statistical analysis of gene expression data identified 3 genes (HSP110, HYOU1 and TCTP) significantly up-regulated in primary tumors of patients who developed lymph node metastasis. We have shown, for the first time, that up-regulation HSP110 and HYOU1 expression is associated with lymph node involvement in CRC. We validated the differences in HSP110 expression in an independent group of 30 patients of all clinical stages by real-time PCR. We identified significant up-regulation of HSP110 expression in colorectal tumors compared to adjacent non-tumoral tissue (p<0.0003). We observed significant differences of HSP110 gene expression between metastatic and localized disease (p=0.031) and negative trend of HSP110 gene expression and overall survival of CRC patients. We suggest that HSP110 gene is a promising molecular predictor in CRC.

Altered expression of miR-21, miR-31, miR-143 and miR-145 is related to clinicopathologic features of colorectal cancer.

OBJECTIVES: Development and metastases of colorectal cancer (CRC) are characterized by multiple genetic alterations. MicroRNAs (miRNAs) are endogenously expressed regulatory noncoding RNAs. Previous, mainly preclinical studies showed altered expression levels of several miRNAs in CRC. METHODS: In our study, the expression levels of miR-21, miR-31, miR-143 and miR-145 in 29 primary colorectal carcinomas and 6 non-tumor adjacent tissue specimens were examined by real-time polymerase chain reaction. miRNA expression levels were also correlated with commonly used clinicopath-ologic features of CRC. RESULTS: Expression levels of analyzed miRNAs significantly differed among tumors and adjacent non-tumor tissues: miR-21 (p = 0.0001) and miR-31 (p = 0.0006) were upregulated, and miR-143 (p = 0.011) and miR-145 (p = 0.003) were downregulated in tumors. For the first time, a high expression of miR-21 was associated with lymph node positivity (p = 0.025) and the development of distant metastases (p = 0.009) in CRC patients. Thus, expression of miR-21 correlated with CRC clinical stage (p = 0.032). Furthermore, tumors >50 mm in maximal tumor diameter were characterized by lower expression of miR-143 (p = 0.006) and miR-145 (p = 0.003). We found no correlation between analyzed miRNAs and serum levels of carcinoembryonic antigen. CONCLUSION: Our results suggest possible roles of miR-21, miR-31, miR-143 and miR-145 in CRC.