Jana Nekvindová

Circulating miR-378 and miR-451 in serum are potential biomarkers for renal cell carcinoma

BackgroundThere is no standard serum biomarker used for diagnosis or early detection of recurrence for renal cell carcinoma (RCC) patients. MicroRNAs (miRNAs) are abundant and highly stable in blood serum, and have been recently described as powerful circulating biomarkers in a wide range of solid cancers. Our aim was to identify miRNA signature that can distinguish the blood serum of RCC patients and matched healthy controls and validate identified miRNAs as potential biomarkers for RCC.MethodsIn the screening phase of the study, blood serum of 15 RCC patients and 12 matched healthy controls were analyzed by use of the TaqMan Low-Density Arrays enabling parallel identification of expression levels of 667 miRNAs through qRT-PCR-based approach. In the validation phase, identified miRNAs were further evaluated on the independent group of 90 RCC patients and 35 matched healthy controls by use of individual qRT-PCR assays and statistically evaluated.ResultsWe identified 30 miRNAs differentially expressed between serum of RCC patients and healthy controls: 19 miRNAs were up-regulated and 11 miRNAs were down-regulated in RCC patients. MiR-378, miR-451 and miR-150 were further evaluated in the independent group of patients, and two of them were successfully validated: levels of miR-378 were increased (p = 0.0003, AUC = 0.71), miR-451 levels were decreased (p < 0.0001, AUC = 0.77) in serum of RCC patients. Combination of miR-378 and miR-451 enable identification of RCC serum with the sensitivity of 81%, specificity 83% and AUC = 0.86.ConclusionsCirculating miRNAs in serum are promising biomarkers in RCC.

Identification and functional screening of microRNAs highly deregulated in colorectal cancer

MicroRNAs (miRNAs) constitute a robust regulatory network with post‐transcriptional regulatory efficiency for almost one half of human coding genes, including oncogenes and tumour suppressors. We determined the expression profile of 667 miRNAs in colorectal cancer (CRC) tissues and paired non‐tumoural tissues and identified 42 differentially expressed miRNAs. We chose miR‐215, miR‐375, miR‐378, miR‐422a and miR‐135b for further validation on an independent cohort of 125 clinically characterized CRC patients and for in vitro analyses. MiR‐215, miR‐375, miR‐378 and miR‐422a were significantly decreased, whereas miR‐135b was increased in CRC tumour tissues. Levels of miR‐215 and miR‐422a correlated with clinical stage. MiR‐135b was associated with higher pre‐operative serum levels of CEA and CA19‐9. In vitro analyses showed that ectopic expression of miR‐215 decreases viability and migration, increases apoptosis and promotes cell cycle arrest in DLD‐1 and HCT‐116 colon cancer cell lines. Similarly, overexpression of miR‐375 and inhibition of miR‐135b led to decreased viability. Finally, restoration of miR‐378, miR‐422a and miR‐375 inhibited G1/S transition. These findings indicate that miR‐378, miR‐375, miR‐422a and miR‐215 play an important role in CRC as tumour suppressors, whereas miR‐135b functions as an oncogene; both groups of miRNA contribute to CRC pathogenesis.

Identification of MicroRNAs associated with early relapse after nephrectomy in renal cell carcinoma patients

Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney. One of the important unmet medical needs in RCC is prognostic biomarker enabling identification of patients at high risk of relapse after nephrectomy. MicroRNAs (miRNAs) constitute a robust regulatory network with posttranscriptional regulatory efficiency for almost one‐half of human coding genes, including oncogenes and tumor suppressors. To identify potential prognostic miRNAs, we analyzed expression profiles in tumors of different prognostic groups of RCC patients. Seventy‐seven patients with clear cell RCC and detailed clinicopathological data were enrolled in a single‐center study. Global miRNA expression profiles were obtained by use of TaqMan Low Density Arrays (754 parallel quantitative reverse‐transcriptase polymerase chain reactions (qRT‐PCR) reactions). For validation of identified miRNAs individual miRNA TaqMan assays were performed in an independent group of patients. We identified tumor relapse‐signature based on the expression of 64 miRNAs differentially expressed between relapse‐free RCC patients and RCC patients who developed relapse (20 miRNAs were increased, 44 miRNAs were decreased). In the validation phase of the study, we successfully confirmed that expression levels of miR‐143, miR‐26a, miR‐145, miR‐10b, miR‐195, and miR‐126 are lower in the tumors of RCC patients who developed tumor relapse, moreover, the lowest levels of these miRNAs we observed in primary metastatic tumors. By using Kaplan–Meier analysis, we identified that miR‐127‐3p, miR‐145, and miR‐126 are significantly correlated with relapse‐free survival of nonmetastatic RCC patients. If further validated, we suggest that identified miRNAs might be used for identification of RCC patients at high risk of early relapse after nephrectomy in clinical practice.

MicroRNA expression profile associated with response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer patients.

BackgroundRectal cancer accounts for approximately one third of all colorectal cancers (CRC), which belong among leading causes of cancer deaths worldwide. Standard treatment for locally advanced rectal cancer (cT3/4 and/or cN+) includes neoadjuvant chemoradiotherapy with fluoropyrimidines (capecitabine or 5-fluorouracil) followed by radical surgical resection. Unfortunately, a significant proportion of tumors do not respond enough to the neoadjuvant treatment and these patients are at risk of relapse. MicroRNAs (miRNAs) are small non-coding RNAs playing significant roles in the pathogenesis of many cancers including rectal cancer. MiRNAs could present the new predictive biomarkers for rectal cancer patients.MethodsWe selected 20 patients who underwent neoadjuvant chemoradiotherapy for advanced rectal cancer and whose tumors were classified as most sensitive or resistant to the treatment. These two groups were compared using large-scale miRNA expression profiling.ResultsExpression levels of 8 miRNAs significantly differed between two groups. MiR-215, miR-190b and miR-29b-2* have been overexpressed in non-responders, and let-7e, miR-196b, miR-450a, miR-450b-5p and miR-99a* have shown higher expression levels in responders. Using these miRNAs 9 of 10 responders and 9 of 10 non-responders (p < 0.05) have been correctly classified.ConclusionsOur pilot study suggests that miRNAs are part of the mechanisms that are involved in response of rectal cancer to the chemoradiotherapy and that miRNAs may be promising predictive biomarkers for such patients. In most miRNAs we identified (miR-215, miR-99a*, miR-196b, miR-450b-5p and let-7e), the connection between their expression and radioresistance or chemoresistance to inhibitors of thymidylate synthetase was already established.

Circulating serum microRNAs as novel diagnostic and prognostic biomarkers for multiple myeloma and monoclonal gammopathy of undetermined significance

Multiple myeloma still remains incurable in the majority of cases prompting a further search for new and better prognostic markers. Emerging evidence has suggested that circulating microRNAs can serve as minimally invasive biomarkers for multiple myeloma and monoclonal gammopathy of undetermined significance. In this study, a global analysis of serum microRNAs by TaqMan Low Density Arrays was performed, followed by quantitative real-time PCR. The analyses revealed five deregulated microRNAs: miR-744, miR-130a, miR-34a, let-7d and let-7e in monoclonal gammopathy of undetermined significance, newly diagnosed and relapsed multiple myeloma when compared to healthy donors. Multivariate logistical regression analysis showed that a combination of miR-34a and let-7e can distinguish multiple myeloma from healthy donors with a sensitivity of 80.6% and a specificity of 86.7%, and monoclonal gammopathy of undetermined significance from healthy donors with a sensitivity of 91.1% and a specificity of 96.7%. Furthermore, lower levels of miR-744 and let-7e were associated with shorter overall survival and remission of myeloma patients. One-year mortality rates for miR-744 and let-7e were 41.9% and 34.6% for the ‘low’ expression and 3.3% and 3.9% for the ‘high’ expression groups, respectively. Median time of remission for both miR-744 and let-7e was approximately 11 months for the ‘low’ expression and approximately 47 months for the ‘high’ expression groups of myeloma patients These data demonstrate that expression patterns of circulating microRNAs are altered in multiple myeloma and monoclonal gammopathy of undetermined significance and miR-744 with let-7e are associated with survival of myeloma patients.

Cervical Microbiota in Women with Preterm Prelabor Rupture of Membranes

Objective To analyze the cervical microbiota in women with preterm prelabor rupture of membranes (PPROM) by pyrosequencing and to document associations between cervical microbiota, cervical inflammatory response, microbial invasion of the amniotic cavity (MIAC), histological chorioamnionitis, and intraamniotic infection (IAI). Study Design Sixty-one women with singleton pregnancies complicated by PPROM were included in the study. Specimens of cervical and amniotic fluid were collected on admission. The cervical microbiota was assessed by 16S rRNA gene sequencing by pyrosequencing. Interleukin (IL)-6 concentration in the cervical fluid and amniotic fluid was measured by ELISA and lateral flow immunoassay, respectively. Results Four bacterial community state types [CST I (Lactobacillus crispatus dominated), CST III (Lactobacillus iners dominated), CST IV-A (non-Lactobacillus bacteria dominated), and CST IV-B (Gardnerella vaginalis and Sneathia sanguinegens dominated)] were observed in the cervical microbiota of women with PPROM. Cervical fluid IL-6 concentrations differed between CSTs (CST I = 145 pg/mL, CST III = 166 pg/mL, CST IV-A = 420 pg/mL, and CST IV-B = 322 pg/mL; p = 0.004). There were also differences in the rates of MIAC, of both MIAC and histological chorioamnionitis, and of IAI among CSTs. No difference in the rate of histological chorioamnionitis was found among CSTs. Conclusions The cervical microbiota in PPROM women in this study was characterized by four CSTs. The presence of non-Lactobacillus CSTs was associated with a strong cervical inflammatory response and higher rates of MIAC, both MIAC and histological chorioamnionitis, and IAI representing a PPROM subtype with pronounced inflammation. CST I represents the dominant type of PPROM with a low rate of MIAC, IAI, and the combination of MIAC and histological chorioamnionitis.

Inhibition of human liver microsomal cytochrome P450 activities by adefovir and tenofovir

Adefovir (PMEA) and tenofovir (PMPA) and their prodrugs, adefovir dipivoxil (bisPOM-PMEA) and tenofovir disoproxil (bisPOC-PMPA), were subjected to a detailed study of their potential to inhibit the activities of human liver microsomal cytochromes P450 (CYP). The inhibition of marker enzyme activities of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 was examined with high-performance liquid chromatography (HPLC) or spectroscopic (fluorescence, luminescence) detection. Adefovir and adefovir dipivoxil did not significantly influence activities of most CYP enzymes. The activity of CYP3A4 was inhibited by adefovir dipivoxil at concentrations over 100 µM. Adefovir and its prodrug inhibited CYP2C9 at concentrations below 100 µM; inhibition by adefovir was of the uncompetitive (at the lower inhibitor concentrations) or of the competitive nature with a Ki = 420 µM. Tenofovir and tenofovir disoproxil influenced the activity of CYP2C9, and competitive inhibition was found with Ki = 580 and 395 µM, respectively. Tenofovir disoproxil was shown to inhibit microsomal CYP2E1 activities by a mixed-type inhibition with Ki values at about 140 µM. The results indicate the possibility of an influence of the compounds tested on the respective CYP activities when used at high doses.

Risk Score based on microRNA expression signature is independent prognostic classifier of glioblastoma patients

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. The prognosis of GBM patients varies considerably and the histopathological examination is not sufficient for individual risk estimation. MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression and were repeatedly proved to play important roles in pathogenesis of GBM. In our study, we performed global miRNA expression profiling of 58 glioblastoma tissue samples obtained during surgical resections and 10 non-tumor brain tissues. The subsequent analysis revealed 28 significantly deregulated miRNAs in GBM tissue, which were able to precisely classify all examined samples. Correlation with clinical data led to identification of six-miRNA signature significantly associated with progression free survival [hazard ratio (HR) 1.98, 95% confidence interval (CI) 1.33-2.94, P < 0.001] and overa+ll survival (HR 2.86, 95% CI 1.91-4.29, P < 0.001). O(6)-methylguanine-DNA methyltransferase methylation status was evaluated as reference method and Risk Score based on six-miRNA signature indicated significant superiority in prediction of clinical outcome in GBM patients. Multivariate Cox analysis indicated that the Risk Score based on six-miRNA signature is an independent prognostic classifier of GBM patients. We suggest that the Risk Score presents promising prognostic algorithm with potential for individualized treatment decisions in clinical management of GBM patients.

Extension of microRNA expression pattern associated with high-risk neuroblastoma

Clinical behavior of neuroblastoma (NBL) is remarkably heterogeneous, as it ranges from spontaneous regression to aggressive clinical phenotype and death. There is increasing body of evidence demonstrating that microRNAs could be considered the potential biomarkers for clinical applications in NBL. In this report, we focus on molecular characterization of high-risk as well as low-risk and intermediate-risk NBL cases in the context of the microRNA expression profile that is specific for the given risk category of the disease. We investigated a total of 30 NBL patients, out of whom there were 19 patients with low- to intermediate-risk and 11 with high-risk NBLs as defined by the Clinical Oncology Group. We determined the expression profiles of 754 microRNAs (miRNAs), whereas the miRNA expression levels were normalized to RNU44, mean expression levels were calculated, and data were analyzed by use of the microarray biostatistical approaches. We identified the signature of 38 miRNAs differentially expressed between these groups of NBL patients (P < 0.05): 17 miRNAs were upregulated and 21 miRNAs were downregulated in the tumors of high-risk NBL patients. We confirm some of the previous observations and we report several new microRNAs associated with aggressive NBL, both being relevant subjects for further translational validation and functional studies.

Aberrant methylation of tumour suppressor genes WT1, GATA5 and PAX5 in hepatocellular carcinoma.

Abstract Background: Aberrant hypermethylation of tumour suppressor genes (TSGs) occurring in hepatocellular carcinoma (HCC) could provide a mean of molecular characterisation of this cancer. The aim of this study was to investigate promoter methylation and gene expression of selected TSGs in HCC to identify candidate genes for further validation as potential biomarkers. Methods: Methylation-specific multiplex ligation-dependent probe amplification method was used to measure the methylation status of 25 TSGs in 49 HCC samples and 36 corresponding non-cancerous liver tissue samples. Relative expression of the differentially methylated genes was assessed at the mRNA level using quantitative PCR. Results: We observed a significantly higher methylation in genes WT1, PAX5, PAX6, PYCARD and GATA5 in HCC compared with control samples. The expression of PAX5 was significantly decreased by methylation; conversely methylation of WT1 was associated with higher mRNA levels. Methylation of GATA5 was significantly associated with overall survival and methylation of WT1 and PAX5 significantly varied between patients with ALBI score 1 vs. 2+3. Moreover, PAX5 was significantly more methylated in patients with tumour grade 2+3 vs. grade 1, and methylation of the PAX5 correlated with the patient’s age at the time of diagnosis. Conclusions: HCC evince aberrant promoter methylation of WT1, PAX5, PAX6, PYCARD and GATA5 genes. Correlation between GATA5, WT1 and PAX5 methylation and clinical/histological parameters is suggestive of applicability of these markers in non-invasive (epi)genetic testing in HCC.