Publications by authors named "Lenka Radova"

86 Publications

Small RNA Sequencing Identifies PIWI-Interacting RNAs Deregulated in Glioblastoma-piR-9491 and piR-12488 Reduce Tumor Cell Colonies .

Front Oncol 2021 13;11:707017. Epub 2021 Aug 13.

Central European Institute of Technology, Masaryk University, Brno, Czechia.

Glioblastoma (GBM) is the most frequently occurring primary malignant brain tumor of astrocytic origin. To change poor prognosis, it is necessary to deeply understand the molecular mechanisms of gliomagenesis and identify new potential biomarkers and therapeutic targets. PIWI-interacting RNAs (piRNAs) help in maintaining genome stability, and their deregulation has already been observed in many tumors. Recent studies suggest that these molecules could also play an important role in the glioma biology. To determine GBM-associated piRNAs, we performed small RNA sequencing analysis in the discovery set of 19 GBM and 11 non-tumor brain samples followed by TaqMan qRT-PCR analyses in the independent set of 77 GBM and 23 non-tumor patients. Obtained data were subsequently bioinformatically analyzed. Small RNA sequencing revealed 58 significantly deregulated piRNA molecules in GBM samples in comparison with non-tumor brain tissues. Deregulation of piR-1849, piR-9491, piR-12487, and piR-12488 was successfully confirmed in the independent groups of patients and controls (all < 0.0001), and piR-9491 and piR-12488 reduced GBM cells' ability to form colonies . In addition, piR-23231 was significantly associated with the overall survival of the GBM patients treated with Stupp regimen ( = 0.007). Our results suggest that piRNAs could be a novel promising diagnostic and prognostic biomarker in GBM potentially playing important roles in gliomagenesis.
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http://dx.doi.org/10.3389/fonc.2021.707017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8415021PMC
August 2021

Complex Interplay of Genes Underlies Invasiveness in Fibrosarcoma Progression Model.

J Clin Med 2021 May 25;10(11). Epub 2021 May 25.

Institute of Biology, Faculty of Medicine in Pilsen, Charles University, 323 00 Plzen, Czech Republic.

Sarcomas are a heterogeneous group of mesenchymal tumours, with a great variability in their clinical behaviour. While our knowledge of sarcoma initiation has advanced rapidly in recent years, relatively little is known about mechanisms of sarcoma progression. JUN-murine fibrosarcoma progression series consists of four sarcoma cell lines, JUN-1, JUN-2, JUN-2fos-3, and JUN-3. JUN-1 and -2 were established from a single tumour initiated in a transgenic mouse, JUN-3 originates from a different tumour in the same animal, and JUN-2fos-3 results from a targeted in vitro transformation of the JUN-2 cell line. The JUN-1, -2, and -3 cell lines represent a linear progression from the least transformed JUN-2 to the most transformed JUN-3, with regard to all the transformation characteristics studied, while the JUN-2fos-3 cell line exhibits a unique transformation mode, with little deregulation of cell growth and proliferation, but pronounced motility and invasiveness. The invasive sarcoma sublines JUN-2fos-3 and JUN-3 show complex metabolic profiles, with activation of both mitochondrial oxidative phosphorylation and glycolysis and a significant increase in spared respiratory capacity. The specific transcriptomic profile of invasive sublines features very complex biological relationships across the identified genes and proteins, with accentuated autocrine control of motility and angiogenesis. Pharmacologic inhibition of one of the autocrine motility factors identified, Ccl8, significantly diminished both motility and invasiveness of the highly transformed fibrosarcoma cell. This progression series could be greatly valuable for deciphering crucial aspects of sarcoma progression and defining new prognostic markers and potential therapeutic targets.
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http://dx.doi.org/10.3390/jcm10112297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8197499PMC
May 2021

Low-burden TP53 mutations in CLL: Clinical impact and clonal evolution within the context of different treatment options.

Blood 2021 May 4. Epub 2021 May 4.

University Hospital Brno, Czech Republic.

Chronic lymphocytic leukemia (CLL) patients with TP53 mutations experience chemo-refractory disease and are therefore indicated for targeted therapy. However, the significance of low-burden TP53 mutations with <10% variant allele frequency (VAF) remains a matter of debate. Here we describe clonal evolution scenarios of low-burden TP53 mutations and analyzed their clinical impact in a "real-world" CLL cohort. TP53 status was assessed by targeted NGS in 511 patients entering first-line treatment with chemo/immunotherapy and 159 relapsed patients treated with targeted agents. Within the pre-therapy cohort, 16% of patients carried low-burden TP53 mutations (0.1-10% VAF). While their presence did not significantly shorten event-free survival after first-line therapy, it affected overall survival (OS). For a subgroup with TP53 mutations of 1-10% VAF, the impact on OS was only observed in patients with unmutated IGHV that had not received targeted therapy, as patients benefited from switching to targeted agents regardless of initial TP53 mutational status. Analysis of the clonal evolution of low-burden TP53 mutations showed that the highest expansion rates were associated with FCR in both first and second-line treatment (median VAF increase 14.8x and 11.8x, respectively) in contrast to treatment with less intense chemo/immunotherapy regimens (1.6x) and without treatment (0.8x). In the relapsed cohort, 33% of patients carried low-burden TP53 mutations, which did not expand significantly upon targeted treatment (median VAF change 1x). Sporadic cases of TP53-mut clonal shifts were connected with the development of resistance-associated mutations. Altogether, our data support the incorporation of low-burden TP53 variants in clinical decision-making.
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http://dx.doi.org/10.1182/blood.2020009530DOI Listing
May 2021

Epithelial to mesenchymal transition and microRNA expression are associated with spindle and apocrine cell morphology in triple-negative breast cancer.

Sci Rep 2021 Mar 4;11(1):5145. Epub 2021 Mar 4.

Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University and University Hospital, 775 15, Olomouc, Czech Republic.

Triple negative breast cancers (TNBC) are a morphologically and genetically heterogeneous group of breast cancers with uncertain prediction of biological behavior and response to therapy. Epithelial to mesenchymal transition (EMT) is a dynamic process characterized by loss of typical epithelial phenotype and acquisition of mesenchymal characteristics. Aberrant activation of EMT can aggravate the prognosis of patients with cancer, however, the mechanisms of EMT and role of microRNAs (miRNAs) in EMT activation is still unclear. The aim of our study was to analyze miRNA expression within areas of TNBCs with cellular morphology that may be related to the EMT process and discuss possible associations. Out of all 3953 re-examined breast cancers, 460 breast cancers were diagnosed as TNBC (11.64%). With regard to complete tumor morphology preservation, the tissue samples obtained from core-cut biopsies and influenced by previous neoadjuvant therapy were excluded. We assembled a set of selected 25 cases to determine miRNA expression levels in relation to present focal spindle cell and apocrine cell morphology within individual TNBCs. We used descriptive (histological typing and morphology), morphometric, molecular (microdissection of tumor and non-tumor morphologies, RNA isolation and purification, microchip analysis) and bioinformatic analysis (including pathway analysis). The results were verified by quantitative real-time PCR (RT-qPCR) on an extended set of 70 TNBCs. The majority of TNBCs were represented by high-grade invasive carcinomas of no special type (NST) with medullary features characterized by well-circumscribed tumors with central necrosis or fibrosis and frequent tendency to spindle-cell and/or apocrine cell transformation. Apocrine and spindle cell transformation showed a specific miRNA expression profile in comparison to other tumor parts, in situ carcinoma or non-tumor structures, particularly down-regulated expression of hsa-miRNA-143-3p and hsa-miRNA-205-5p and up-regulated expression of hsa-miR-22-3p, hsa-miRNA-185-5p, and hsa-miR-4443. Apocrine cell tumor morphology further revealed decreased expression of hsa-miR-145-5p and increased expression of additional 14 miRNAs (e.g. hsa-miR-182-5p, hsa-miR-3135b and hsa-miR-4417). Pathway analysis for target genes of these miRNAs revealed several shared biological processes (i.e. Wnt signaling, ErbB signaling, MAPK signaling, endocytosis and axon guidance), which may in part contribute to the EMT and tumor progression. We provide the first miRNA expression profiling of specific tissue morphologies in TNBC. Our results demonstrate a specific miRNA expression profile of apocrine and spindle cell morphology which can exhibit a certain similarity with the EMT process and may also be relevant for prognosis and therapy resistance of TNBC.
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http://dx.doi.org/10.1038/s41598-021-84350-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933252PMC
March 2021

Functional analysis of germline mutation causing inherited thrombocytopenia and secondary acute lymphoblastic leukemia or essential thrombocythemia.

Platelets 2021 Aug 21;32(6):838-841. Epub 2020 Aug 21.

Center of Molecular Medicine, Central European Institute of Technology, Masaryk University (CEITEC MU), Brno, Czech Republic.

Germline mutations in gene cause inherited thrombocytopenia with leukemia predisposition. Here, we report on functional validation of W380R mutation segregating with thrombocytopenia in a family where two family members also suffered from acute lymphoblastic leukemia (ALL) or essential thrombocythemia (ET). analysis predicted impaired DNA binding due to W380R mutation. Functional analysis showed that this mutation prevents the ETV6 protein from localizing into the cell nucleus and impairs the transcriptional repression activity of ETV6. Based on the germline mutation, ET probably started with somatic V617F mutation, whereas ALL could be caused by diverse mechanisms: high-hyperdiploidity; somatic deletion of exon 1 gene; or somatic mutations of other genes found by exome sequencing of the ALL sample taken at the diagnosis.
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http://dx.doi.org/10.1080/09537104.2020.1802416DOI Listing
August 2021

Hepatocellular carcinoma: Gene expression profiling and regulation of xenobiotic-metabolizing cytochromes P450.

Biochem Pharmacol 2020 07 13;177:113912. Epub 2020 Mar 13.

Department of Clinical Biochemistry and Diagnostics, University Hospital Hradec Kralove, Czech Republic. Electronic address:

Hepatocellular carcinoma (HCC) remains a highly prevalent and deadly disease, being among the top causes of cancer-related deaths worldwide. Despite the fact that the liver is the major site of biotransformation, studies on drug metabolizing enzymes in HCC are scarce. It is known that malignant transformation of hepatocytes leads to a significant alteration of their metabolic functions and overall deregulation of gene expression. Advanced stages of the disease are thus frequently associated with liver failure, and severe alteration of drug metabolism. However, the impact of dysregulation of metabolic enzymes on therapeutic efficacy and toxicity in HCC patients is largely unknown. Here we demonstrate a significant down-regulation in European Caucasian patients of cytochromes P450 (CYPs), the major xenobiotic-metabolizing enzymes, in HCC tumour samples as compared to their surrounding non-cancerous (reference) tissue. Moreover, we report for the first time the association of the unique CYP profiles with specific transcriptome changes, and interesting correlations with expression levels of nuclear receptors and with the histological grade of the tumours. Integrated analysis has suggested certain co-expression profiles of CYPs with lncRNAs that need to be further characterized. Patients with large tumours with down-regulated CYPs could be more vulnerable to drug toxicity; on the other hand, such tumours would eliminate drugs more slowly and should be more sensitive to pharmacotherapy (except in the case of pro-drugs where activation is necessary).
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http://dx.doi.org/10.1016/j.bcp.2020.113912DOI Listing
July 2020

Novel genetic variant of HPS1 gene in Hermansky-Pudlak syndrome with fulminant progression of pulmonary fibrosis: a case report.

BMC Pulm Med 2019 Oct 16;19(1):178. Epub 2019 Oct 16.

Department of Internal Medicine, Hematology and Oncology, University Hospital and Faculty of Medicine, Jihlavská 20, 625 00, Brno, Czech Republic.

Background: Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder that is associated with oculocutaneous albinism, bleeding diathesis, granulomatous colitis, and highly penetrant pulmonary fibrosis in some subtypes. Homozygous or compound heterozygous pathological variants in HPS1, HPS3, HPS4, and several other genes lead to clinical manifestation of the disease.

Case Presentation: A 57-year-old female was admitted with congenital oculocutaneous albinism, thrombocytopathy and late-onset accelerated pulmonary fibrosis (first symptoms from age 50 onwards). Chest high-resolution computed tomography identified thickening of peribronchovascular interstitium, bronchiectasis, reticulations, honeycombing, ground glass opacities and lung parenchyma consolidations. HPS was clinically suspected. We performed whole exome sequencing (WES), a form of massive parallel sequencing, of proband-parents trio. Whole exome libraries were processed using KAPA Hyper Prep Kit, SeqCap EZ MedExome Enrichment Kit and HyperCap Bead Kit according to the SeqCap EZ HyperCap Workflow. The paired-end 2 × 75 bp sequencing was performed on the Illumina NextSeq 500 Sequencer (Illumina Inc., USA). Furthermore, obtained variants by WES were evaluated using a virtual panel of genes: HPS1, AP3B1, HPS3, HPS4, HPS5, HPS6, DTNBP1, BLOC1S3, and PLDN. We identified a compound heterozygous genotype in HPS1 gene in the proband. We identified a pathogenic frameshift variant c.1189delC; p.(Gln397Serfs*2), resulting in a premature stop codon. This variant has been previously associated with HPS. Furthermore, we characterized previously undescribed nonsense variant c.1507C > T; p.(Gln503*), resulting in a premature stop codon and mRNA degradation through nonsense-mediated decay. Sanger sequencing validated the presence of both variants and simultaneously confirmed the heterozygous carrier status of parents. Unfortunately, the patient died due to fulminant progression of pulmonary fibrosis 2 months after diagnostics.

Conclusions: Compound heterozygous mutations in HPS1 in the proband lead to disruption of HPS1 gene and clinical manifestation of HPS with severe pulmonary fibrosis. This case illustrates the need to consider HPS in differential diagnostics of pulmonary fibrosis. Pulmonary fibrosis is a common cause of death in HPS patients. Earlier diagnosis may enable better treatment for these patients.
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http://dx.doi.org/10.1186/s12890-019-0941-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794755PMC
October 2019

Cerebrospinal Fluid MicroRNA Signatures as Diagnostic Biomarkers in Brain Tumors.

Cancers (Basel) 2019 Oct 12;11(10). Epub 2019 Oct 12.

Department of Neurosurgery, University Hospital Brno, Brno 625 00, Czech Republic.

Central nervous system (CNS) malignancies include primary tumors that originate within the CNS as well as secondary tumors that develop as a result of metastatic spread. Circulating microRNAs (miRNAs) were found in almost all human body fluids including cerebrospinal fluid (CSF), and they seem to be highly stable and resistant to even extreme conditions. The overall aim of our study was to identify specific CSF miRNA patterns that could differentiate among brain tumors. These new biomarkers could potentially aid borderline or uncertain imaging results onto diagnosis of CNS malignancies, avoiding most invasive procedures such as stereotactic biopsy or biopsy. In total, 175 brain tumor patients (glioblastomas, low-grade gliomas, meningiomas and brain metastases), and 40 non-tumor patients with hydrocephalus as controls were included in this prospective monocentric study. Firstly, we performed high-throughput miRNA profiling (Illumina small RNA sequencing) on a discovery cohort of 70 patients and 19 controls and identified specific miRNA signatures of all brain tumor types tested. Secondly, validation of 9 candidate miRNAs was carried out on an independent cohort of 105 brain tumor patients and 21 controls using qRT-PCR. Based on the successful results of validation and various combination patterns of only 5 miRNA levels (miR-30e, miR-140, let-7b, mR-10a and miR-21-3p) we proposed CSF-diagnostic scores for each tumor type which enabled to distinguish them from healthy donors and other tumor types tested. In addition to this primary diagnostic tool, we described the prognostic potential of the combination of miR-10b and miR-196b levels in CSF of glioblastoma patients. In conclusion, we performed the largest study so far focused on CSF miRNA profiling in patients with brain tumors, and we believe that this new class of biomarkers have a strong potential as a diagnostic and prognostic tool in these patients.
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http://dx.doi.org/10.3390/cancers11101546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826583PMC
October 2019

Profiling of biological and environmental risk factors in immunogenetic subgroups of chronic lymphocytic leukemia - Czech national study.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2020 Dec 26;164(4):425-434. Epub 2019 Sep 26.

Department of Internal Medicine - Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic.

Aims: This is a nation-wide survey of chronic lymphocytic leukemia (CLL) patients at six large hematology centers in the Czech Republic. The aim was to identify specific populations, social, and health characteristics of CLL subgroups divided according to the immunogenetic features of their B cell receptors (BCRs) and clonality.

Patients And Methods: Questionnaires directed to specific health, social, and environmental conditions were collected in a cohort of 573 CLL patients. For these patients, immunoglobulin heavy chain gene rearrangements were also analyzed in order to gain information about their clonality, IGHV mutational status, and the presence of stereotyped BCRs. Data extracted from the questionnaires were analyzed statistically in the context of immunogenetic features of the cohort.

Results: There were no statistically significant differences in the data collected in the survey between patients with mutated and patients with unmutated IGHV. However, patients with oligoclonal CLL reported health conditions such as hypercholesterolemia, hypertension, herpes simplex, tumors, and also, separately, CLL in 1 degree relatives, more often than their monoclonal counterparts. In patients with stereotyped BCRs, we found more frequent alcohol consumption and gastric infections in subset #1 cases and frequent cholecystectomies and familial CLL in subset #2 cases.

Conclusion: To the best of our knowledge, this study is the first to investigate CLL immunogenetic features and clonality in the context of epidemiological data. We reported statistically significant associations suggesting the influence of certain health and social conditions on a number of clonal populations expanding in CLL and also on characteristic BCR features, especially stereotypy.
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http://dx.doi.org/10.5507/bp.2019.046DOI Listing
December 2020

MiR-376b-3p Is Associated With Long-term Response to Sunitinib in Metastatic Renal Cell Carcinoma Patients.

Cancer Genomics Proteomics 2019 Sep-Oct;16(5):353-359

Masaryk Memorial Cancer Institute, Department of Comprehensive Cancer Care, Faculty of Medicine, Masaryk University, Brno, Czech Republic

Background/aim: Sunitinib is a tyrosine kinase inhibitor routinely used as first-line therapy in metastatic renal cell carcinoma (mRCC). Emerging evidence suggests that microRNAs (miRNAs) could be suitable biomarkers with predictive potential in mRCC. The aim of this study was to identify miRNA-based predictive biomarkers of therapy response to avoid unnecessary therapy to non-responding patients.

Patients And Methods: High-throughput miRNA microarray profiling was performed on a cohort of 47 patients treated with sunitinib. Validation of candidate miRNAs was carried out on an independent cohort of 132 mRCC patients using qRT-PCR.

Results: Out of 158 miRNAs (65 down-regulated, 93 up-regulated), six miRNAs were chosen for independent validation and miR-376b-3p was confirmed to be differentially expressed in tumors of patients with primary resistance versus long-term response (p<0.0002).

Conclusion: A predictive miRNA associated with progression-free survival in metastatic renal cell carcinoma patients treated with sunitinib was identified.
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http://dx.doi.org/10.21873/cgp.20140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727077PMC
January 2020

Bending of DNA duplexes with mutation motifs.

DNA Res 2019 Aug;26(4):341-352

CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic.

Mutations can be induced by environmental factors but also arise spontaneously during DNA replication or due to deamination of methylated cytosines at CpG dinucleotides. Sites where mutations occur with higher frequency than would be expected by chance are termed hotspots while sites that contain mutations rarely are termed coldspots. Mutations are permanently scanned and repaired by repair systems. Among them, the mismatch repair targets base pair mismatches, which are discriminated from canonical base pairs by probing altered elasticity of DNA. Using biased molecular dynamics simulations, we investigated the elasticity of coldspots and hotspots motifs detected in human genes associated with inherited disorders, and also of motifs with Czech population hotspots and de novo mutations. Main attention was paid to mutations leading to G/T and A+/C pairs. We observed that hotspots without CpG/CpHpG sequences are less flexible than coldspots, which indicates that flexible sequences are more effectively repaired. In contrary, hotspots with CpG/CpHpG sequences exhibited increased flexibility as coldspots. Their mutability is more likely related to spontaneous deamination of methylated cytosines leading to C > T mutations, which are primarily targeted by base excision repair. We corroborated conclusions based on computer simulations by measuring melting curves of hotspots and coldspots containing G/T mismatch.
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http://dx.doi.org/10.1093/dnares/dsz013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704406PMC
August 2019

Bioinformatic pipelines for whole transcriptome sequencing data exploitation in leukemia patients with complex structural variants.

PeerJ 2019 12;7:e7071. Epub 2019 Jun 12.

Central European Institute of Technology, Masaryk University, Brno, Czech Republic.

Background: Extensive genome rearrangements, known as chromothripsis, have been recently identified in several cancer types. Chromothripsis leads to complex structural variants (cSVs) causing aberrant gene expression and the formation of fusion genes, which can trigger cancer development, or worsen its clinical course. The functional impact of cSVs can be studied at the RNA level using whole transcriptome sequencing (total RNA-Seq). It represents a powerful tool for discovering, profiling, and quantifying changes of gene expression in the overall genomic context. However, bioinformatic analysis of transcriptomic data, especially in cases with cSVs, is a complex and challenging task, and the development of proper bioinformatic tools for transcriptome studies is necessary.

Methods: We designed a bioinformatic workflow for the analysis of total RNA-Seq data consisting of two separate parts (pipelines): The first pipeline incorporates a statistical solution for differential gene expression analysis in a biologically heterogeneous sample set. We utilized results from transcriptomic arrays which were carried out in parallel to increase the precision of the analysis. The second pipeline is used for the identification of fusion genes. Special attention was given to the filtering of false positives (FPs), which was achieved through consensus fusion calling with several fusion gene callers. We applied the workflow to the data obtained from ten patients with chronic lymphocytic leukemia (CLL) to describe the consequences of their cSVs in detail. The fusion genes identified by our pipeline were correlated with genomic break-points detected by genomic arrays.

Results: We set up a novel solution for differential gene expression analysis of individual samples and fusion gene detection from total RNA-Seq data. The results of the differential gene expression analysis were concordant with results obtained by transcriptomic arrays, which demonstrates the analytical capabilities of our method. We also showed that the consensus fusion gene detection approach was able to identify true positives (TPs) efficiently. Detected coordinates of fusion gene junctions were in concordance with genomic breakpoints assessed using genomic arrays.

Discussion: Byapplying our methods to real clinical samples, we proved that our approach for total RNA-Seq data analysis generates results consistent with other genomic analytical techniques. The data obtained by our analyses provided clues for the study of the biological consequences of cSVs with far-reaching implications for clinical outcome and management of cancer patients. The bioinformatic workflow is also widely applicable for addressing other research questions in different contexts, for which transcriptomic data are generated.
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http://dx.doi.org/10.7717/peerj.7071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6571010PMC
June 2019

High-throughput analysis revealed mutations' diverging effects on exon 7 splicing.

RNA Biol 2019 10 19;16(10):1364-1376. Epub 2019 Jun 19.

Medical Genomics RG, Central European Institute of Technology, Masaryk University , Brno , Czech Republic.

Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the gene's exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5'ss) and 5'ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5'ss mutations potentiated the use of three different cryptic 5'ss. Generally, single mutations supporting cryptic 5'ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5'ss. Analyzing double mutants supported the predominating splicing regulatory elements' effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5'ss can be one of the main factors driving cryptic 5'ss use.
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http://dx.doi.org/10.1080/15476286.2019.1630796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779402PMC
October 2019

Identification of microRNA signatures in umbilical cord blood associated with maternal characteristics.

PeerJ 2019 30;7:e6981. Epub 2019 May 30.

Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic.

Background: Umbilical cord blood could serve as useful source of blood markers enabling more efficient and reliable prenatal and neonatal diagnostics. MicroRNAs (miRNAs) are ubiquitous in body fluids where they were used for detecting and monitoring various physiological and pathological conditions. In this descriptive study, we aimed to identify changes in miRNA expression profiles associated with basic maternal somatic and epidemiological characteristics.

Methods: Study is based on 24 mothers from the Pilot phase of CELSPAC: TNG (Central European Longitudinal Studies of Parents and Children: The Next Generation) study. Cord blood was collected at time of delivery and global miRNA profiling was performed using microRNA Ready-to-use PCR Human Panel I+II TaqMan microarrays. Expression profiles were statistically evaluated in relation to maternal age, BMI, pregnancy weight gain, blood type, Rh factor status, allergies during pregnancy, addictive substance abuse and smoking status.

Results: We analyzed expression of 752 human mature miRNAs in 24 samples of umbilical cord blood. For all maternal characteristics tested we described a specific signature of significantly deregulated miRNAs ( < 0.05). Analysis revealed seven miRNA associated with maternal age (three increased and four decreased in women younger than 35 years), 14 miRNAs associated with BMI status (five miRNAs increased and nine miRNAs decreased in women with BMI > 25) and nine miRNAs associated with maternal weight gain during pregnancy (eight miRNAs increased, and one miRNA decreased in women with weight gain < 12 kg). Additionally, 17 miRNAs correlated to blood type (two miRNAs decreased in blood type A, 11 increased in blood type B, two miRNAs increased in blood type AB and two miRNAs increased in blood type 0) and 17 miRNAs to Rh status of mother. We also detected seven miRNAs deregulated in umbilical cord blood of women with allergy (four increased and three decreased in women with allergy), four miRNAs associated to addictive substance abuse status (two up- and two downregulated in women with addictive substance abuse) and eight miRNAs associated with maternal cigarette smoking during pregnancy.

Conclusions: We successfully described differences in miRNA profiles in umbilical cord blood associated with basic characteristics connected with mother. Our data suggest that miRNAs in umbilical cord blood are detectable and associated with a wide range of maternal characteristics. These results indicate that miRNAs could potentially serve, and should be studied, as biomarkers for screening and diagnosis of pregnancy-associated complications and pathologies.
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http://dx.doi.org/10.7717/peerj.6981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545228PMC
May 2019

A novel germline mutation of the gene in familial interstitial pneumonia.

Hum Genome Var 2019 5;6:12. Epub 2019 Mar 5.

2Central European Institute of Technology, Masaryk University, Brno, Czech Republic.

Different genes related to alveolar stability have been associated with familial interstitial pneumonia (FIP). Here, we report a novel, rare variant in a family with idiopathic interstitial pneumonia (IIP). We performed whole-exome sequencing on germline DNA samples from four members of one family; three of them showed signs of pulmonary fibrosis (idiopathic interstitial pneumonia) with autosomal-dominant inheritance. A heterozygous single nucleotide variant c.532 G > A in the gene has been identified. This variant encodes the substitution p.(Val178Met), localized within the carbohydrate recognition domain of surfactant protein A and segregates with the genes causing idiopathic interstitial pneumonia. This rare variant has not been previously reported. We also analyzed the detected sequence variant in the protein structure . The replacement of valine by the larger methionine inside the protein may cause a disruption in the protein structure. The c.532 G > A variant was further validated using Sanger sequencing of the amplicons, confirming the diagnosis in all symptomatic family members. Moreover, this variant was also found by Sanger sequencing in one other symptomatic family member and one young asymptomatic family member. The autosomal-dominant inheritance, the family history of IIP, and the evidence of a mutation occurring in part of the gene all suggest a novel variant that causes FIP.
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http://dx.doi.org/10.1038/s41439-019-0044-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399245PMC
March 2019

MicroRNA-15b-5p Predicts Locoregional Relapse in Head and Neck Carcinoma Patients Treated With Intensity-modulated Radiotherapy.

Cancer Genomics Proteomics 2019 Mar-Apr;16(2):139-146

Central European Institute of Technology, Masaryk University, Brno, Czech Republic

Background/aim: Head and neck cancers are a heterogenous group of epithelial tumors represented mainly by squamous cell carcinomas (HNSCC), which are the sixth most common type of cancer worldwide. Surgery together with radiotherapy (RT) is among the basic treatment modalities for most HNSCC patients. Various biomarkers aiming to predict patients' response to RT are currently investigated. The reason behind this effort is, on one hand, to distinguish radioresistant patients that show weak benefit from RT and, on the other hand, reduce the ionizing radiation dose in less aggressive radiosensitive HNSCC with possibly less acute or late toxicity.

Materials And Methods: A total of 94 HNSCC patients treated by definitive intensity-modulated radiotherapy were included in our retrospective study. We used a global expression analysis of microRNAs (miRNAs) in 43 tumor samples and validated a series of selected miRNAs in an independent set of 51 tumors.

Results: We identified miR-15b-5p to be differentially expressed between patients with short and long time of locoregional control (LRC). Kaplan-Meier analysis confirmed that HNSCC patients with higher expression of miR-15b-5p reach a significantly longer locoregional relapse-free survival compared to patients expressing low levels. Finally, multivariable Cox regression analysis revealed that miR-15b-5p is an independent predictive biomarker of LRC in HNSCC patients (HR=0.25; 95% CI=0.05-0.78; p<0.016).

Conclusion: miR-15b-5p represents a potentially helpful biomarker for individualized treatment decisions concerning the management of HNSCC patients.
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http://dx.doi.org/10.21873/cgp.20119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6489689PMC
June 2019

ATM and TP53 mutations show mutual exclusivity but distinct clinical impact in mantle cell lymphoma patients.

Leuk Lymphoma 2019 06 10;60(6):1420-1428. Epub 2019 Jan 10.

a Department of Internal Medicine - Hematology and Oncology , University Hospital Brno and Faculty of Medicine, Masaryk University , Brno , Czech Republic.

Mantle cell lymphoma (MCL) is characterized by the hallmark t(11;14)(q13;q32) translocation, leading to cyclin D1 over-expression. Additionally, disrupting the DNA damage response pathway through or defects plays an important role in MCL pathogenesis. Using deep next-generation sequencing we analyzed the mutual composition of and mutations in 72 MCL patients, and assessed their impact on progression-free survival (PFS) and overall survival (OS). Mutated and alleles were found in 51% (37/72) and 22% (16/72) of the cases examined, respectively, with only three patients harboring mutations in both genes. Only a mutated gene was associated with the significantly reduced PFS and OS and the same output was observed when and defective groups included also sole deletions 11q and 17p, respectively. Determining the exact ATM/p53 pathway dysfunction may influence the selection of MCL patients for innovative therapies based on the targeted inhibition of selected proteins.
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http://dx.doi.org/10.1080/10428194.2018.1542144DOI Listing
June 2019

Tumor expression of miR-34a-3p is an independent predictor of recurrence in non-muscle-invasive bladder cancer and promising additional factor to improve predictive value of EORTC nomogram.

Urol Oncol 2019 03 13;37(3):184.e1-184.e7. Epub 2018 Nov 13.

Central European Institute of Technology, Masaryk University, Brno, Czech Republic; Department of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute, Brno, Czech Republic. Electronic address:

Objectives: Non-muscle-invasive bladder cancer (NMIBC) is a heterogeneous disease characterized by a high primary tumor recurrence rate. Current prognostic systems used for predicting recurrence in individual patients have limitations and do not consider the biological background of this tumor type. Our study aimed to find microRNAs (miRNAs) associated with NMIBC recurrence.

Methods: Seventy-eight NMIBC patients were prospectively enrolled and divided into exploratory and validation cohorts. Out of these patients, 32 developed recurrence within 18 months after surgery, while 46 did not show any sign of recurrence after 30 months. Expression profiles of 2,578 miRNAs were obtained using Affymetrix miRNA microarrays and candidate miRNAs validated using the individual quantitative reverse-transcription polymerase chain reaction (qRT-PCR).

Results: The expression profiling revealed a set of 137 miRNAs differentially expressed between NMIBC patients with and without recurrence (P < 0.05). In the validation phase, miR-34a-3p had a significantly higher expression in tumors of NMIBC patients without recurrence (P = 0.0155). Decreased expression of miR-34a-3p was associated with significantly shorter recurrence-free survival (P = 0.009). Cox regression analysis confirmed that miR-34a-3p is an independent biomarker associated with a lower risk of recurrence (hazard ratio (HR) = 0.3184, 95% confidence interval = 0.003-0.681, P = 0.0258). Combination of miR-34a-3p and European Organization for Research and Treatment of Cancer risk score into one predictive model enabled to predict individual risk of recurrence with high statistical significance and analytical performance (P < 0.0001; area under curve = 0.8368; sensitivity 83%, and specificity 75%).

Conclusions: Our data suggest that miR-34a-3p is an independent biomarker of NMIBC recurrence and a promising candidate for further independent validations as an additional factor to improve predictive value of European Organization for Research and Treatment of Cancer nomogram.
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http://dx.doi.org/10.1016/j.urolonc.2018.10.014DOI Listing
March 2019

Activation-induced deaminase and its splice variants associate with trisomy 12 in chronic lymphocytic leukemia.

Ann Hematol 2019 Feb 27;98(2):423-435. Epub 2018 Oct 27.

Central European Institute of Technology, Center of Molecular Medicine, Masaryk University, Kamenice 5/A35, 625 00, Brno, Czech Republic.

Activation-induced cytidine deaminase (AID) is a mutator enzyme essential for somatic hypermutation (SHM) and class switch recombination (CSR) during effective adaptive immune responses. Its aberrant expression and activity have been detected in lymphomas, leukemias, and solid tumors. In chronic lymphocytic leukemia (CLL) increased expression of alternatively spliced AID variants has been documented. We used real-time RT-PCR to quantify the expression of AID and its alternatively spliced transcripts (AIDΔE4a, AIDΔE4, AIDivs3, and AIDΔE3E4) in 149 CLL patients and correlated this expression to prognostic markers including recurrent chromosomal aberrations, the presence of complex karyotype, mutation status of the immunoglobulin heavy chain variable gene, and recurrent mutations. We report a previously unappreciated association between higher AID transcript levels and trisomy of chromosome 12. Functional analysis of AID splice variants revealed loss of their activity with respect to SHM, CSR, and induction of double-strand DNA breaks. In silico modeling provided insight into the molecular interactions and structural dynamics of wild-type AID and a shortened AID variant closely resembling AIDΔE4, confirming its loss-of-function phenotype.
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http://dx.doi.org/10.1007/s00277-018-3520-5DOI Listing
February 2019

A novel germline mutation in GP1BA gene N-terminal domain in monoallelic Bernard-Soulier syndrome.

Platelets 2018 Dec 17;29(8):827-833. Epub 2018 Oct 17.

a Department of Internal Medicine- Hematology and Oncology, University Hospital Brno and Faculty of Medicine , Masaryk University , Brno , Czech Republic.

Mutations in the GP1BA gene have been associated with platelet-type von Willebrand disease and Bernard-Soulier syndrome. Here, we report a novel GP1BA mutation in a family with autosomal dominant macrothrombocytopenia and mild bleeding. We performed analyses of seven family members. Using whole-exome sequencing of germline DNA samples, we identified a heterozygous single-nucleotide change in GP1BA (exone2:c.176T>G), encoding a p.Leu59Arg substitution in the N-terminal domain, segregating with macrothrombocytopenia. This variant has not been previously reported. We also analysed the structure of the detected sequence variant in silico. In particular, we used the crystal structure of the human platelet receptor GP Ibα N-terminal domain. Replacement of aliphatic amino-acid Leu 59 with charged, polar and larger arginine probably disrupts the protein structure. An autosomal dominant mode of inheritance, a family history of mild bleeding episodes, aggregation pattern in affected individuals together with evidence of mutation occurring in part of the GP1BA gene encoding the leucine-rich repeat region suggest a novel variant causing monoallelic Bernard-Soulier syndrome.
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http://dx.doi.org/10.1080/09537104.2018.1529300DOI Listing
December 2018

Circulating exosomal long noncoding RNA PRINS-First findings in monoclonal gammopathies.

Hematol Oncol 2018 Dec 13;36(5):786-791. Epub 2018 Sep 13.

Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.

Multiple myeloma is the second most common hematological malignancy characterized by focal lesions of malignant plasma cells in the bone marrow. These lesions contain subclones that directly influence survival of patients. Bone marrow biopsies are single-site biopsies and thus cannot contain all information about the tumor. In contrast, liquid biopsies analyze circulating cells and molecules that are secreted from all sites of the tumor. Long noncoding RNA molecules are one class of these molecules. We performed a two-phase biomarker study investigating lncRNA expression profiles in exosomes of peripheral blood serum of newly diagnosed multiple myeloma (MM) patients, monoclonal gammopathy of undetermined significance (MGUS) patients in comparison with healthy donors (HD). Surprisingly, this analysis revealed dysregulation of only one exosomal lncRNA PRINS in MM vs HD. Overall, MM and MGUS patients were distinguished from HD with sensitivity of 84.9% and specificity of 83.3%. Our study suggests a possible diagnostic role for exosomal lncRNA PRINS in monoclonal gammopathies patients.
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http://dx.doi.org/10.1002/hon.2554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585732PMC
December 2018

MicroRNA miR-34a downregulates FOXP1 during DNA damage response to limit BCR signalling in chronic lymphocytic leukaemia B cells.

Leukemia 2019 02 15;33(2):403-414. Epub 2018 Aug 15.

Molecular Medicine, CEITEC MU, Brno, Czech Republic.

The variable clinical course in chronic lymphocytic leukaemia (CLL) largely depends on p53 functionality and B-cell receptor (BCR) signalling propensity; however, it is unclear if there is any crosstalk between these pathways. We show that DNA damage response (DDR) activation leads to down-modulating the transcriptional factor FOXP1, which functions as a positive BCR signalling regulator and its high levels are associated with worse CLL prognosis. We identified microRNA (miRNA) miR-34a as the most prominently upregulated miRNA during DDR in CLL cells in vitro and in vivo during FCR therapy (fludarabine, cyclophosphamide, rituximab). MiR-34a induced by DDR activation and p53 stabilization potently represses FOXP1 expression by binding in its 3'-UTR. The low FOXP1 levels limit BCR signalling partially via derepressing BCR-inhibitory molecule CD22. We also show that low miR-34a levels can be used as a biomarker for worse response or shorter progression free survival in CLL patients treated with FCR chemoimmunotherapy, and shorter overall survival, irrespective of TP53 status. Additionally, we have developed a method for the absolute quantification of miR-34a copies and defined precise prognostic/predictive cutoffs. Overall, herein, we reveal for the first time that B cells limit their BCR signalling during DDR by down-modulating FOXP1 via DDR-p53/miR-34a axis.
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http://dx.doi.org/10.1038/s41375-018-0230-xDOI Listing
February 2019

C-terminal RUNX1 mutation in familial platelet disorder with predisposition to myeloid malignancies.

Int J Hematol 2018 Dec 6;108(6):652-657. Epub 2018 Aug 6.

Central European Institute of Technology (CEITEC), University Hospital, Masaryk University, Brno, Czech Republic.

Here we report a C-terminal RUNX1 mutation in a family with platelet disorder and predisposition to myeloid malignancies. We identified the mutation c.866delG:p.Gly289Aspfs*22 (NM_001754) (RUNX1 b-isoform NM_001001890; c.785delG:p.Gly262Aspfs*22) using exome sequencing of samples obtained from eight members of a single family. The mutation found in our pedigree is within exon eight and the transactivation domain of RUNX1. One of the affected individuals developed myelodysplastic syndrome (MDS), which progressed to acute myelogenous leukemia (AML). A search for the second hit which led to the development of MDS and later AML in this individual revealed the PHF6 gene variant (exon9:c.872G > A:p.G291E; NM_001015877), BCORL1 (exon3:c.1111A > C:p.T371P; NM_001184772) and BCOR gene variant (exon4:c.2076dupT:p.P693fs; NM_001123383), which appear to be very likely second hits participating in the progression to myeloid malignancy.
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http://dx.doi.org/10.1007/s12185-018-2514-3DOI Listing
December 2018

Circulating PIWI-Interacting RNAs piR-5937 and piR-28876 Are Promising Diagnostic Biomarkers of Colon Cancer.

Cancer Epidemiol Biomarkers Prev 2018 09 5;27(9):1019-1028. Epub 2018 Jul 5.

Centre for Molecular Medicine, Central European Institute of Technology, Masaryk University, Brno, Czech Republic.

The early detection of colon cancer is one of the main prerequisites for successful treatment and mortality reduction. Circulating PIWI-interacting RNAs (piRNA) were recently identified as novel promising biomarkers. The purpose of the study was to assess the profiles of piRNAs in blood serum of colon cancer patients with the aim to identify those with high diagnostic potential. Blood serum samples from 403 colon cancer patients and 276 healthy donors were included in this 3-phase biomarker study. Large-scale piRNA expression profiling was performed using Illumina small RNA sequencing. The diagnostic potential of selected piRNAs was further validated on independent training and validation sets of samples using RT-qPCR. In total, 31 piRNAs were found to be significantly deregulated in serum of cancer patients compared with healthy donors. Based on the levels of piR-5937 and piR-28876, it was possible to differentiate between cancer patients and healthy donors with high sensitivity and specificity. Moreover, both piRNAs exhibited satisfactory diagnostic performance also in patients with stage I disease and enabled detection of colon cancer with higher sensitivity than currently used biomarkers CEA and CA19-9. Finally, the expression of analyzed piRNAs in blood restored significantly 1 month after the surgical resection. Based on our findings, piRNAs are abundant in human blood serum. Furthermore, their levels in colon cancer have been observed to be significantly deregulated. However, their involvement in carcinogenesis must be further established. piRNAs could serve as promising noninvasive biomarkers for early detection of colon cancer. .
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http://dx.doi.org/10.1158/1055-9965.EPI-18-0318DOI Listing
September 2018

Identification of microRNAs differentially expressed in glioblastoma stem-like cells and their association with patient survival.

Sci Rep 2018 02 12;8(1):2836. Epub 2018 Feb 12.

Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic.

Glioblastoma stem-like cells (GSCs) are critical for the aggressiveness and progression of glioblastoma (GBM) and contribute to its resistance to adjuvant treatment. MicroRNAs (miRNAs) are small, non-coding RNAs controlling gene expression at the post-transcriptional level, which are known to be important regulators of the stem-like features. Moreover, miRNAs have been previously proved to be promising diagnostic biomarkers in several cancers including GBM. Using global expression analysis of miRNAs in 10 paired in-vitro as well as in-vivo characterized primary GSC and non-stem glioblastoma cultures, we identified a miRNA signature associated with the stem-like phenotype in GBM. 51 most deregulated miRNAs classified the cell cultures into GSC and non-stem cell clusters and identified a subgroup of GSC cultures with more pronounced stem-cell characteristics. The importance of the identified miRNA signature was further supported by demonstrating that a Risk Score based on the expression of seven miRNAs overexpressed in GSC predicted overall survival in GBM patients in the TCGA dataset independently of the IDH1 status. In summary, we identified miRNAs differentially expressed in GSCs and described their association with GBM patient survival. We propose that these miRNAs participate on GSC features and could represent helpful prognostic markers and potential therapeutic targets in GBM.
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http://dx.doi.org/10.1038/s41598-018-20929-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809429PMC
February 2018

Genome-wide identification of urinary cell-free microRNAs for non-invasive detection of bladder cancer.

J Cell Mol Med 2018 03 24;22(3):2033-2038. Epub 2018 Jan 24.

Department of Urologic Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic.

Urinary microRNAs (miRNAs) are emerging as clinically useful tool for early and non-invasive detection of various types of cancer including bladder cancer (BCA). In this study, 205 patients with BCA and 99 healthy controls were prospectively enrolled. Expression profiles of urinary miRNAs were obtained using Affymetrix miRNA microarrays (2578 miRNAs) and candidate miRNAs further validated in independent cohorts using qRT-PCR. Whole-genome profiling identified 76 miRNAs with significantly different concentrations in urine of BCA compared to controls (P < 0.01). In the training and independent validation phase of the study, miR-31-5p, miR-93-5p and miR-191-5p were confirmed to have significantly higher levels in urine of patients with BCA in comparison with controls (P < 0.01). We further established 2-miRNA-based urinary DxScore (miR-93-5p, miR-31-5p) enabling sensitive BCA detection with AUC being 0.84 and 0.81 in the training and validation phase, respectively. Moreover, DxScore significantly differed in the various histopathological subgroups of BCA and decreased post-operatively. In conclusion, we identified and independently validated cell-free urinary miRNAs as promising biomarkers enabling non-invasive detection of BCA.
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http://dx.doi.org/10.1111/jcmm.13487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824364PMC
March 2018

Comprehensive Group Therapy of Obesity and Its Impact on Selected Anthropometric and Postural Parameters.

Cent Eur J Public Health 2017 Dec;25(4):326-331

Centre for Research and Science, Faculty of Health Sciences, Palacký University Olomouc, Olomouc, Czech Republic.

Objective: Obesity is a multifactorial disease. This non-infectious epidemic has reached pandemic proportions in the 21 century. Posture is a dynamic process referring to an active maintenance of body movement segments against the action of external forces. The aim of the study was to investigate the effect of comprehensive group therapy for obese persons on selected anthropometric and postural parameters.

Methods: The study comprised 53 females with a mean age of 44.5 years (range 29–65 years, standard deviation 9.42 years, median 44 years), who completed a controlled weight loss programme. At the beginning and at the end of the programme, anthropometric parameters (Body Mass Index (BMI), weight and waist circumference) were measured and the posturography tests Limits of Stability (LOS) and Motor Control Test (MCT) were performed using the NeuroCom's SMART EquiTest system. The data were statistically analyzed using R software at a level of significance of 0.05.

Results: There were positive changes after the controlled weight loss programme in anthropometric parameters (BMI reduction, with p<0.001; waist circumference reduction, with p<0.001; and weight loss, with p<0.001), postural stability with statistically significant (p<0.05) improvements in both postural activity (LOS test parameters) and reactions (MCT parameters).

Conclusion: The study showed a statistically significant effect of comprehensive group therapy for obesity in terms of reductions in waist circumference, body weight and BMI, and thus the overall reduction of both cardiovascular and metabolic risks, as well as improved postural skills (activity and reactions).
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http://dx.doi.org/10.21101/cejph.a4780DOI Listing
December 2017

MiR-215-5p is a tumor suppressor in colorectal cancer targeting EGFR ligand epiregulin and its transcriptional inducer HOXB9.

Oncogenesis 2017 Dec 4;6(11):399. Epub 2017 Dec 4.

Central European Institute of Technology, Masaryk University, Brno, Czech Republic.

Growing evidence suggests that microRNAs are involved in the development and progression of colorectal cancer (CRC). In the present study, deregulation and functioning of tumor-suppressive miR-215-5p was evaluated in CRC. In total, 448 tumor tissues and 325 paired adjacent healthy tissues collected from Czech and Spain cohorts of CRC patients have been used for miR-215-5p expression analyses. A series of in vitro experiments have been performed using transient transfection of miR-215-5p mimics into four CRC cell lines to identify specific cellular processes affected by miR-215-5p. Further, the effects of miR-215-5p on tumor growth were evaluated in vivo using NSG mice and stable cell line overexpressing miR-215-5p. Target mRNAs of miR-215-5p were tested using luciferase assay and western blot analyses. We found that miR-215-5p is significantly downregulated in tumor tissues compared with non-tumor adjacent tissues and its decreased levels correlate with the presence of lymph node metastases, tumor stage, and shorter overall survival in CRC patients. Overexpression of miR-215-5p significantly reduced proliferation, clonogenicity, and migration of CRC cells, lead to cell cycle arrest in G2/M phase and p53-dependent induction of apoptosis. The ability of miR-215-5p to inhibit tumor growth was confirmed in vivo. Finally, we confirmed epiregulin and HOXB9 to be the direct targets of miR-215-5p. As epiregulin is EGFR ligand and HOXB9 is its transcriptional inducer, we suggest that the main molecular link between miR-215-5p and CRC cells phenotypes presents the EGFR signaling pathway, which is one of the canonical pathogenic pathways in CRC.
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http://dx.doi.org/10.1038/s41389-017-0006-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5868056PMC
December 2017
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