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.

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© F e r r a t a S t o r t i F o u n d a t i o nclassification. 22 In this study, a new serum miRNAs expression profile, potent enough to distinguish newly diagnosed MM and MGUS patients from healthy controls, was created based on TaqMan Low Density Arrays (TLDA). This profile was validated by quantitative realtime PCR (qPCR) on a larger cohort of newly diagnosed and relapsed MM as well as MGUS patients. Moreover, miRNAs levels were correlated with clinical, biochemical and cytogenetic characteristics and survival data. Methods Patients and healthy donorsPeripheral blood (PB) serum samples from 103 newly diagnosed MM patients, 18 MM patients in relapse, 57 MGUS and 30 healthy donors (HD) from the Faculty Hospital Brno, Czech Republic, were obtained for this study. PB serum samples were collected as follows: centrifugation 3500 rpm/15 min/20°C. Samples were frozen as 0.5 mL aliquots, stored at -80°C and thawed only once. For 70 MM and 36 MGUS samples, BMPCs were obtained for routine interphase fluorescence in situ hybridization analysis (I-FISH), as described previously. 23 Patients' and donors' characteristics are described in Table 1 and in the Online Supplementary Table S1. For 6 newly diagnosed MM patients, BMPCs and exosomal and non-exosomal fraction from PB serum were collected. All patients signed an informed consent form approved by the hospital ethical committee before enrollment into this study. MiRNA extractionTotal RNA enriched for miRNAs was extracted from all serum samples using miRNeasy Kit (Qiagen) modified for circulating miRNAs according to the manufacturer's instructions. MiRNA/RNA quantity was assessed on a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific) as measurement of each sample 2 times with mean SD=0.292 ng/μL. All samples fit into the Nanodrop ND-1000 validated measuring range. Exosomes precipitationExosomes were collected using ExoQuick Exosome Precipitation Solution (System Biosciences). Serum samples were centrifuged for 3500 rpm/10 min/4°C, 250 μL of serum was combined with 63 μL of ExoQuick, incubated for 30 min/4°C and centrifuged for 2 min/13000 rpm. Exosomal and non-exosomal fraction was used for miRNA/RNA extraction, as described above. TaqMan Low Density ArraysMegaplex profiling using human TaqMan Low Density miRNA Arrays A+B, v3.0 (TLDA) (Life Technologies) was performed to evaluate the expression of 667 miRNA (see Online Supplementary Methods). QPCR was performed on the ABI7900HT system; raw data were analyzed using SDS software v.2.3, RQ Manager v1.2.1 (Life Technologies). Candidate miRNA confirmation by qPCR and quantification of miRNAIndividual TaqMan miRNA assays for 6 miRNA (hsa-miR-222-002276, hsa-miR-744-002324, hsa-miR-130a-000454, hsa-miR34a-000426, hsa-let-7e-002406, hsa-let-7d-002283, Life Technologies) were used for qPCR on a 7500 Real-Time PCR System. QPCR and reverse transcription was performed following the manufacturer´s recommendations (see Online Supplementary Methods). Absolute quantification to determine the copy number of each miRNA per 1...

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.

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.

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.

The Chaetosphaeriaceae are a diverse group of pigmented, predominantly phialidic hyphomycetes comprised of several holomorphic genera including Chaetosphaeria, the most prominent genus of the family. Although the morphology of the teleomorphs of the majority of Chaetosphaeria is rather uniform, their associated anamorphs primarily exhibit the variability and evolutionary change observed in the genus. An exception from the morphological monotony among Chaetosphaeria species is a group characterised by scolecosporous, hyaline to light pink, multiseptate, asymmetrical ascospores and a unique three-layered ascomatal wall. Paragaeumannomyces sphaerocellularis, the type species of the genus, exhibits these morphological traits and is compared with similar Chaetosphaeria with craspedodidymum- and chloridium-like synanamorphs. Morphological comparison and phylogenetic analyses of the combined ITS-28S sequences of 35 isolates and vouchers with these characteristics revealed a strongly-supported, morphologically well-delimited clade in the Chaetosphaeriaceae containing 16 species. The generic name Paragaeumannomyces is applied to this monophyletic clade; eight new combinations and five new species, i.e. P. abietinussp. nov., P. eleganssp. nov., P. granulatussp. nov., P. sabinianussp. nov. and P. smokiensissp. nov., are proposed. A key to Paragaeumannomyces is provided. Using morphology, cultivation studies and phylogenetic analyses of ITS and 28S rDNA, two additional new species from freshwater and terrestrial habitats, Codinaea paniculatasp. nov. and Striatosphaeria castaneasp. nov., are described in the family. A codinaea-like anamorph of S. castanea forms conidia with setulae at each end in axenic culture; this feature expands the known morphology of Striatosphaeria. A chaetosphaeria-like teleomorph is experimentally linked to Dendrophoma cytisporoides, a sporodochial hyphomycete and type species of Dendrophoma, for the first time.

Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney accounting for about 3 % of adult malignancies. MicroRNAs (miRNAs) are a class of naturally occurring, short non-coding RNAs that regulate gene expression at the post-transcriptional level. We determined global miRNA expression profiles of RCC and parallel renal parenchyma tissues by using quantitative reverse transcriptase-polymerase chain reaction-based TaqMan low-density arrays. Afterward, we validated the difference in miR-210 expression levels on the larger group of RCC patients (35 RCC versus 10 non-tumorous parenchyma samples). Functional in vitro experiments were performed on ACHN and CAKI-2 RCC cell lines transfected with miRNA-210 inhibitor. Cell viability, apoptosis, cell cycle, scratch wound migration assay, and invasion assay (xCELLigence) were performed. We have identified original ccRCC-specific miRNA signature in clinical samples (73 miRNAs were significantly downregulated and five miRNAs upregulated (P < 0.003)). Increased expression levels of miR-210 in RCC tumor tissue were independently validated. We observed decreased viability of ACHN and CAKI-2 cells and accumulation of CAKI-2 in G2 phase of cell cycle after silencing of miR-210 expression. Downregulation of miR-210 also reduced the migratory and invasive potential of ACHN metastatic RCC cells. Moreover, we showed downregulation of HIF1a protein in both cell lines after miR-210 silencing indicating participation of miR-210 in hypoxic processes of RCC not only through regulation of its target mRNAs but also by indirect regulation of HIF1a. To our knowledge, this is the first report to show miR-210 regulatory effects on cell migration, invasive potential, and HIF1a protein in RCC cells.