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miRNA expression in triple-negative breast cancers (TNBC) has mainly been studied from a methodological viewpoint. However, it has not been considered that miRNA expression profile may be associated with a specific morphological entity inside every tumor. The verification of this hypothesis on a set of 25 TNBCs was the subject of our previous work, where we confirmed specific expression of the studied miRNAs in 82 samples of different morphologies including inflammatory infiltrate, spindle cell, clear cell, and metastases after RNA extraction and purification as well as microchip and biostatistical analysis. In the current work, we demonstrate a low suitability of in situ hybridization method for miRNA detection compared to RT-qPCR, and in detail discuss the biological role of 8 miRNAs with the most significant changes of expression.

Background/aim: The monoclonal antibody bevacizumab is a standard drug used in combination with oxaliplatin (FOLFOX) or irinotecan (FOLFIRI) based chemotherapy in the first or second-line treatment of metastatic colorectal cancer (mCRC). Our previous study identified and subsequently validated 4 microRNAs in a small group of patients as predictors of the therapeutic response to bevacizumab combined with chemotherapy. The aim of this follow-up study is to confirm the predictive ability of these tissue miRNAs in a larger independent cohort of mCRC patients., Patients and Methods: The retrospective study included 92 patients with generalized-radically inoperable tumors treated with the combined therapy of bevacizumab/FOLFOX in a standard regimen., Results: Expression levels of candidate miRNA biomarkers (miR-92b-3p, miR-3156-5p, miR-10a-5p and miR-125a-5p) were determined in tumor tissue specimens and statistically evaluated. MiR-92b-3p and miR-125a-5p were confirmed to be associated with radiological response according to RECIST criteria (p=0.005 and 0.05, respectively) and to be up-regulated in responders to bevacizumab/FOLFOX therapy. Higher levels of miR-92b-3p were also significantly associated with extended progression-free survival (p=0.024)., Conclusion: We have successfully confirmed miR-92b-3p to be up-regulated in tumor tissue of mCRC patients with good response to bevacizumab/FOLFOX therapy., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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.

Background/aim: Rectal cancer accounts for approximately one-third of all colorectal cancers. Currently, the standard treatment for locally advanced rectal cancer (LARC) is neoadjuvant chemoradiotherapy (CRT) with capecitabine or 5-fluorouracil followed by curative surgery. Unfortunately, only 20% of patients with LARC present complete pathological response after CRT, whereas in 20-40% cases the response is poor or absent. The aim of our study was to evaluate whether microRNAs (miRNAs) in tumor biopsy specimen have the potential to predict therapeutic response in LARC patients., Patients and Methods: In total 87 LARC patients treated by CRT were enrolled in our prospective study. To identify predictive miRNAs, we used small RNA sequencing in 40 tumor biopsy samples of LARC patients (20 responders, 20 non-responders) and qPCR validation of selected miRNA candidates., Results: In the discovery phase of the study, we identified 69 miRNAs to have significantly different expression between the group of responders (TRG 1,2) and a group of non-responders (TRG 4,5) to neoadjuvant CRT. Among these miRNAs, 48 showed a lower expression and 21 showed higher expression in tumor tissues from poorly responding LARC patients. Five miRNAs were selected for validation, but only miR-487a-3p was confirmed to have a significantly higher expression in the tumor biopsy specimens of non-responders to neoadjuvant CRT (p<0.0006, AUC=0.766). Gene Ontology (GO) clustering and pathway enrichment analysis of the miR-487a-3p mRNA targets, revealed potential mechanisms behind miR-487a-3p roles in chemoradioresistance (e.g. TGF-beta signaling pathway, protein kinase activity, double-stranded DNA binding, or microRNAs in cancer)., Conclusion: By combination of miRNA expression profiling and integrative computational biology we identified miR-487a-3p as a potential predictive biomarker of CRT response in LARC patients., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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

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., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background/aim: In Western countries, most patients with gastric cancer (GC) present in advanced stages. Therefore, there is imminent clinical need for novel diagnostic and prognostic biomarkers. Deregulation of microRNAs has been reported as a frequent event in GC development in a number of studies. Our study validated the potential of microRNAs to serve as diagnostic and prognostic biomarkers in patients with GC from the Central European population., Materials and Methods: Using quantitative real-time polymerase chain reaction, expression levels of six microRNAs (miR-10b, -21, -93, -107, - 143, and -145) were examined in 67 tumor tissues and 67 paired adjacent gastric tissues, and correlated with clinicopathological features of GC patients., Results: Expression levels of miR-10b, miR-21, miR-93, and miR-107 were significantly higher in GC samples compared to non-tumor tissue. Furthermore, the expression levels of miR-10b, miR-143, and miR-145 positively correlated with advanced stages, and increased expression of miR-10b, miR-21 and miR-145 was significantly associated with worse prognosis of gastric cancer patients., Conclusion: Our results indicate that selected tissue microRNAs have the potential to serve as relevant diagnostic and prognostic biomarkers of GC in a central European population., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Targeted therapy with the tyrosine kinase inhibitor sunitinib is used in the first line of metastatic renal cell carcinoma (mRCC) treatment. The aim of the present study was independent validation of microRNAs (miRNAs) identified in previous studies as biomarkers predicting response to sunitinib therapy., Materials and Methods: Based on a literature search, 10 miRNAs were chosen from six relevant studies as candidates for validation: miR-155, miR-484, miR-221, miR-222, miR-425, miR-133, miR-410, miR-141, miR-628 and miR-942. Validation of these miRNAs was performed on cohort of 56 patients with mRCC with extremely good or poor response responses to sunitinib treatment using quantitative reverse transcription-polymerase chain reaction. Patients were divided into either responding (n=24) or non-responding (n=32) groups to sunitinib treatment according to Response Evaluation Criteria in Solid Tumors and progression-free survival (PFS). All patients in the responding group had PFS longer than 18 months, PFS of non-responders was shorter than 6 months in all cases., Results: miR-942 and miR-133 were confirmed as being differentially expressed in tumors of responding and non-responding patients. It was not possible to validate the predictive value of other tested miRNAs, however, expression of miR-221 and miR-425 tended to be positively associated with therapeutic response (p<0.1). We further developed a model based on the combination of miR-942 and miR-133 expression, that enabled identification of non-responding patients with mRCC with sensitivity of 78% and specificity of 79% (area under the curve=0.8071)., Conclusion: Following further independent validation, detection of these miRNAs may prevent unnecessary and costly approaches to therapy in non-responding patients with mRCC., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Renal cell carcinoma (RCC) accounts for 2-3% of all malignant tumours. Metastatic RCC (mRCC) is commonly treated with tyrosine kinase inhibitors (TKI). Effective TKIs administration can be achieved only by accurate prediction of therapeutical response. Therefore, the aim of this study was to analyse papers concerning predictive potential of microRNA (miRNA)., Material and Methods: We chose seven candidate miRNAs and analysed their expression on 44 patients divided into cohort with poor and good response to sunitinib treatment. Patients were divided into two groups according to progression-free survival. RNA from tissue samples was isolated and expression of selected miRNAs was measured using quantitative PCR with miRNA-specific TaqMan probes., Results: We successfully validated two miRNAs to be differentially expressed in responding and non-responding patients to sunitinib treatment. Other analysed miRNAs have not shown predictive potential., Conclusion: From miRNAs studied so far, two miRNAs had predictive value according to present study.Key words: microRNA - renal cell carcinoma - sunitib 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. Supported by Ministry of Health of the Czech Republic, grant No. 15-34678A. All rights reserved.Submitted: 19. 3. 2018Accepted: 20. 3. 2018.

Purpose: miR-196b-5p has been previously implicated in malignant transformation; however, its role in colorectal cancer has not been fully explored. In this study, we examine the clinical and biological relevance of miR-196b-5p, and the molecular pathways regulated by miR-196b-5p in colorectal cancer. Experimental Design: miR-196b-5p expression was quantitated by qRT-PCR in 2 independent cohorts composed of 292 patients with colorectal cancer in total, to explore its biomarker potential. Transient and stable gain- and loss-of-function experiments were conducted in a panel of colorectal cancer cell lines and mice, to evaluate the impact of miR-196b-5p on proliferation, chemosensitivity, migration/invasion, and metastases formation in vitro and in vivo The molecular pathways influenced by miR-196b-5p were characterized using whole transcriptome profiling, in silico target prediction tools, luciferase interaction assays, and phenocopy/rescue gene knockdown experiments. Results: Low miR-196b-5p expression was significantly associated with metastases and poor outcomes in 2 independent colorectal cancer patient cohorts ( P < 0.05, log-rank test). miR-196b-5p inhibition led to significantly increased colorectal cancer cell migration/invasion and metastases formation in mice, whereas ectopic overexpression showed the opposite phenotype. Molecular profiling and target confirmation identified an interaction between miR-196b-5p and HOXB7 and GALNT5, which in turn regulated colorectal cancer cell migration. Conclusions: The association of low levels of miR-196b-5p and poor prognosis in patients with colorectal cancer can be explained by its influence on cancer cell migration and metastases formation. miR-196b-5p has an impact on colorectal cancer progression pathways through direct interaction with genes involved in cancer cell migration. Clin Cancer Res; 23(17); 5255-66. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Introduction: Urinary microRNAs (miRNAs) are emerging as a clinically useful tool for early and non-invasive detection of various types of cancer. The aim of this study was to evaluate whether let-7 family miRNAs differ in their urinary concentrations between renal cell carcinoma (RCC) cases and healthy controls., Materials and Methods: In the case-control study, 69 non-metastatic clear-cell RCC patients and 36 gender/age-matched healthy controls were prospectively enrolled. Total RNA was purified from cell-free supernatant of the 105 first morning urine specimens. Let-7 family miRNAs were determined in cell-free supernatant using quantitative miRNA real-time reverse-transcription PCR and absolute quantification approach., Results: Concentrations of all let-7 miRNAs (let-7a, let-7b, let-7c, let-7d, let-7e and let-7g) were significantly higher in urine samples obtained from RCC patients compared to healthy controls (P < 0.001; P < 0.001; P = 0.005; P = 0.006; P = 0.015 and P = 0.002, respectively). Subsequent ROC analysis has shown that let-7a concentration possesses good ability to differentiate between cases and controls with area under curve being 0.8307 (sensitivity 71%, specificity 81%)., Conclusions: We have shown that let-7 miRNAs are abundant in the urine samples of patients with clear-cell RCC, and out of six let-7 family members, let-7a outperforms the others and presents promising non-invasive biomarker for the detection of RCC., Competing Interests: Potential conflict of interests: None declared.

Purpose: Characterization of colorectal cancer transcriptome by high-throughput techniques has enabled the discovery of several differentially expressed genes involving previously unreported miRNA abnormalities. Here, we followed a systematic approach on a global scale to identify miRNAs as clinical outcome predictors and further validated them in the clinical and experimental setting. Experimental Design: Genome-wide miRNA sequencing data of 228 colorectal cancer patients from The Cancer Genome Atlas dataset were analyzed as a screening cohort to identify miRNAs significantly associated with survival according to stringent prespecified criteria. A panel of six miRNAs was further validated for their prognostic utility in a large independent validation cohort ( n = 332). In situ hybridization and functional experiments in a panel of colorectal cancer cell lines and xenografts further clarified the role of clinical relevant miRNAs. Results: Six miRNAs (miR-92b-3p, miR-188-3p, miR-221-5p, miR-331-3p, miR-425-3p, and miR-497-5p) were identified as strong predictors of survival in the screening cohort. High miR-188-3p expression proves to be an independent prognostic factor [screening cohort: HR = 4.137; 95% confidence interval (CI), 1.568-10.917; P = 0.004; validation cohort: HR = 1.538; 95% CI, 1.107-2.137; P = 0.010, respectively]. Forced miR-188-3p expression increased migratory behavior of colorectal cancer cells in vitro and metastases formation in vivo ( P < 0.05). The promigratory role of miR-188-3p is mediated by direct interaction with MLLT4, a novel identified player involved in colorectal cancer cell migration. Conclusions: miR-188-3p is a novel independent prognostic factor in colorectal cancer patients, which can be partly explained by its effect on MLLT4 expression and migration of cancer cells. Clin Cancer Res; 23(5); 1323-33. ©2016 AACR ., (©2016 American Association for Cancer Research.)

MicroRNAs (miRNAs) have been proven to be important oncogenes and tumor suppressors in wide range of cancers, including renal cell carcinoma (RCC). In our study, we evaluated miRNA-429 as potential diagnostic/prognostic biomarker in 172 clear cell RCC patients and as a potential regulator of epithelial-mesenchymal transition (EMT) in vitro. We demonstrated that miR-429 is down-regulated in tumor tissue samples (P < 0.0001) and is significantly associated with cancer metastasis (P < 0.0001), shorter disease-free (P = 0.0105), and overall survival (P = 0.0020). In addition, ectopic expression of miR-429 in 786-0 RCC cells followed by TGF-β treatment led to increase in the levels of E-cadherin expression (P < 0.0001) and suppression of cellular migration (P < 0.0001) in comparison to TGF-β-treated controls. Taken together, our findings suggest that miR-429 may serve as promising diagnostic and prognostic biomarker in RCC patients. We further suggest that miR-429 has a capacity to inhibit loss of E-cadherin in RCC cells undergoing EMT and consequently attenuate their motility.

Early detection of colorectal cancer is the main prerequisite for successful treatment and reduction of mortality. Circulating microRNAs were previously identified as promising diagnostic, prognostic and predictive biomarkers. The purpose of this study was to identify serum microRNAs enabling early diagnosis and prognosis prediction of colon cancer. In total, serum samples from 427 colon cancer patients and 276 healthy donors were included in three-phase biomarker study. Large-scale microRNA expression profiling was performed using Illumina small RNA sequencing. Diagnostic and prognostic potential of identified microRNAs was validated on independent training and validation sets of samples using RT-qPCR. Fifty-four microRNAs were found to be significantly deregulated in serum of colon cancer patients compared to healthy donors (P < 0.01). A diagnostic four-microRNA signature consisting of miR-23a-3p, miR-27a-3p, miR-142-5p and miR-376c-3p was established (AUC = 0.917), distinguishing colon cancer patients from healthy donors with sensitivity of 89% and specificity of 81% (AUC = 0.922). This panel of microRNAs exhibited high diagnostic performance also when analyzed separately in colon cancer patients in early stages of the disease (T1-4N0M0; AUC = 0.877). Further, a prognostic panel based on the expression of miR-23a-3p and miR-376c-3p independent of TNM stage was established (HR 2.30; 95% CI 1.44-3.66; P < 0.0004). In summary, highly sensitive signatures of circulating microRNAs enabling non-invasive early detection and prognosis prediction of colon cancer were identified., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: In metastatic colorectal cancer (mCRC), panitumumab is generally considered to be ineffective after the progression on cetuximab therapy. However, few studies have demonstrated that a small subset of mCRC patients may benefit from panitumumab in this setting., Patients and Methods: In our study, wild-type KRAS mCRC patients, enrolled into the nationwide Czech registry CORECT between January 2007 and December 2012, were screened for panitumumab therapy after progression on cetuximab., Results: We identified 26 mCRC in the registry with well documented progression on cetuximab in combination with irinotecan-based chemotherapy (FOLFIRI or irinotecan alone) who received panitumumab monotherapy. Partial response (PR) was achieved in 3 (11.5%) patients and stable disease (SD) in 7 (26.9%) patients after 8 weeks of therapy. Thirteen (50.0%) patients had evidence of progressive disease (PD) and in 3 (11.5%) cases response was not available. Furthermore, we confirmed that higher expression levels of newly described biomarker, miR-31-5p, in tumor are significantly associated with shorter progression-free survival (PFS) in patients treated with cetuximab (p=0.038); however, we did not observe association between miR-31-5p and response to panitumumab in mCRC patients after progression on cetuximab., Conclusion: It remains possible that a subset of mCRC patients may benefit from panitumumab after progression on cetuximab., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

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

Background: Primary tumor spread to the liver is the major cause of disease progression and death in patients with colorectal cancer (CRC). MicroRNAs (miRNAs) are small non-coding RNAs that are involved in cancer development and progression, but their role in metastasis has not been extensively investigated., Materials and Methods: Firstly, expression profiling of 752 miRNAs in 20 primary tumors and their corresponding liver metastases was performed. Secondly, validation of the results was carried out on an independent cohort of 66 patients with metastatic CRC using reverse transcription-quantitative polymerase chain (RT-qPCR) reaction., Results: In total, 33 miRNAs were found to be significantly deregulated in liver metastases compared to their primary tumors. Fifteen miRNAs were chosen for subsequent validation, which confirmed significantly reduced expression of miR-143, miR 10b, and miR-28-5p, and increased expression of miR 122, miR-122*, and miR 885-5p in the tissue of liver metastases., Conclusion: These results indicate that miRNAs could serve as new therapeutic targets in patients with metastatic CRC., (Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.)

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor. Despite radical surgery and radiotherapy supported by chemotherapy, the disease still remains incurable with an extremely low median survival rate of 12-15 months from the time of initial diagnosis. The main cause of treatment failure is considered to be the presence of cells that are resistant to the treatment. MicroRNAs (miRNAs) as regulators of gene expression are involved in the tumor pathogenesis, including GBM. MiR-338 is a brain-specific miRNA which has been described to target pathways involved in proliferation and differentiation. In our study, miR-338-3p and miR-338-5p were differentially expressed in GBM tissue in comparison to non-tumor brain tissue. Overexpression of miR-338-3p with miRNA mimic did not show any changes in proliferation rates in GBM cell lines (A172, T98G, U87MG). On the other hand, pre-miR-338-5p notably decreased proliferation and caused cell cycle arrest. Since radiation is currently the main treatment modality in GBM, we combined overexpression of pre-miR-338-5p with radiation, which led to significantly decreased cell proliferation, increased cell cycle arrest, and apoptosis in comparison to irradiation-only cells. To better elucidate the mechanism of action, we performed gene expression profiling analysis that revealed targets of miR-338-5p being Ndfip1, Rheb, and ppp2R5a. These genes have been described to be involved in DNA damage response, proliferation, and cell cycle regulation. To our knowledge, this is the first study to describe the role of miR-338-5p in GBM and its potential to improve the sensitivity of GBM to radiation.

Esophageal cancer is a malignant disease with poor prognosis, increasing incidence, and ineffective treatment options. MicroRNAs are post-transcriptional regulators of gene expression involved in many biological processes including carcinogenesis. We determined miR-205 expression levels in tumor/non-tumor tissues of 45 esophageal cancer patients using qPCR and found that decreased level of miR-205 in tumor tissue correlates with poor overall survival in esophageal adenocarcinoma patients. Further, we observed significantly higher levels of miR-205 in tumor tissue of esophageal squamous cell carcinoma. Ectopic overexpression of miR-205 in adenocarcinoma cell line SK-GT-4 led to decreased cell proliferation, cell cycle arrest in G1, and decreased migration ability. Conversely, in squamous cell line KYSE-150, same effects like inhibition of proliferation, migration, and colony-forming potential and cell cycle arrest in G2 were observed after silencing of miR-205. We performed global gene expression profiling and revealed that suppressive functioning of miR-205 in adenocarcinoma could be realized through regulation of epithelial-mesenchymal transition (EMT), whereas oncogenic in squamous cell carcinoma by regulation of metalloproteinase 10. Our results suggest that miR-205 could serve as biomarker in esophageal cancer and acts as a tumor suppressor in esophageal adenocarcinoma and oncogene in esophageal squamous cell carcinoma.

The aim of our study was to investigate whether microRNAs (miRNAs) could serve as predictive biomarkers to anti-EGFR therapy (cetuximab, panitumumab) in patients with RAS wild-type (wt-RAS) metastatic colorectal cancer (mCRC). Historical cohort of 93 patients with mCRC (2006-2009) was included and further divided into exploratory and validation cohorts. MiRNAs expression profiling was performed on the exploratory cohort of 41 wt-KRAS mCRC patients treated with cetuximab to identify miRNAs associated with time to progression (TTP). The validation was performed on two independent cohorts: 28 patients of wt-RAS mCRC treated with cetuximab and 24 patients of wt-RAS mCRC treated with panitumumab. We identified 9 miRNAs with significantly different expression between responders and non-responders to cetuximab therapy (P ≤ 0.01). These 9 miRNAs were further evaluated in two independent cohorts of patients and miR-31-3p (P < 0.001) and miR-31-5p (P < 0.001) were successfully confirmed as strongly associated with TTP in wt-RAS mCRC patients treated with cetuximab but not panitumumab. When evaluated on the complete cohort of cetuximab patients (N = 69), miR-31-3p (HR, 5.10; 95% CI, 2.52-10.32; P < 0.001) and miR-31-5p (HR, 4.80; 95% CI, 2.50-9.24; P < 0.001) were correlated with TTP on the comparable level of significance. There was no difference in miR-31-5p/3p expression levels in RAS mutated and wild-type tumor samples. MiR-31-5p/3p are promising predictive biomarkers of cetuximab response in wt-RAS mCRC patients.

Serum microRNAs are emerging as a clinically useful tool for early and non-invasive detection of various cancer types including renal cell carcinoma (RCC). Based on our previous results, we performed the study to analyze circulating serum miR-378 and miR-210 in patients with various histological subtypes of RCC. RNA was purified from blood serum samples of 195 RCC patients and 100 healthy controls. The levels of miR-378 and miR-210 in serum were determined absolutely using quantitative real-time PCR. Pre- and postoperative levels of both microRNAs were compared in 20 RCC patients. Significantly increased serum levels of both miR-378 and miR-210 enabled to clearly distinguish RCC patients and healthy controls with 80% sensitivity and 78% specificity if analyzed in combination (p<0.0001), and their levels significantly decreased in the time period of three months after radical nephrectomy (p<0.0001). Increased level of miR-378 positively correlates with disease-free survival (p=0.036) and clinical stage (p=0.0476). The analysis of serum miR-378 and miR-210 proved their potential to serve as powerful non-invasive diagnostic and prognostic biomarkers in RCC.

Esophageal adenocarcinoma (EAC) is highly aggressive malignancy that frequently develops from Barrett's esophagus (BE), a premalignant pathologic change occurring in the lower end of the esophagus. MicroRNAs (miRNAs) are small, non-coding RNAs that function as posttranscriptional regulators of gene expression and were repeatedly proved to play key roles in pathogenesis of BE as well as EAC. In our study, we used Affymetrix GeneChip miRNA arrays to obtain miRNA expression profiles in total of 119 tissue samples [24 normal esophageal mucosa (EM), 60 BE and 35 EAC]. We identified a number of miRNAs, that showed altered expression progressively in sequence EM, BE and EAC, including for instance miR-21, miR-25, miR-194 and miR-196a with increasing levels (P < 0.0015) and miR-203, miR-205, miR-210 and miR-378 with decreasing levels (P < 0.0001). The subsequent analysis revealed four diagnostic miRNA signatures enabling to distinguish EM and BE [12 miRNAs, area under curve (AUC) = 0.971], EM and EAC (13 miRNAs, AUC = 1.0), BE without and BE with dysplasia (21 miRNAs, AUC = 0.856) and BE without dysplastic changes and BE with dysplasia together with EAC (2 miRNAs, AUC = 0.886). We suggest that miRNA expression profiling expands current knowledge in molecular pathology of Barrett's-based carcinogenesis and enables identification of molecular biomarkers for early detection of BE dysplasia and progression to EAC., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Esophageal cancer is the malignant tumor with very poor prognosis and increasing incidence often diagnosed at very late stage, so the prognosis of affected patients is unsatisfactory, despite the development of therapeutic option such as surgery, chemotherapy and radiotherapy. Consequently, there is a great need for biomarkers to allow a tailored multimodality approach with increased efficiency. Altered expression of microRNAs has been reported in wide range of malignancies, including esophageal cancer. The aim of this study was to examine the expression levels of candidate microRNAs in esophageal cancer and evaluate their diagnostic and prognostic potential., Findings: Using quantitative real-time PCR, expression levels of 9 candidate microRNAs were examined in 62 tissue samples, 23 esophageal adenocarcinomas, 22 esophageal squamous cell carcinomas and 17 adjacent esophageal mucosa samples. MicroRNA expression levels were further analyzed in regards to clinico-pathological features of esophageal cancer patients. We observed significantly decreased levels of miR-203 and increased levels of miR-21 in adenocarcinoma tissues when compared to normal mucosa. MiR-29c and miR-148 indicated good ability to distinguish between histological subtypes of esophageal cancer. MiR-203 and miR-148 were linked to disease-free survival and overall survival in esophageal adenocarcinoma patients, and miR-148 also in esophageal squamous cell carcinoma patients., Conclusions: Our data suggest that altered expression of miR-21, miR-29c, miR-148 and miR-203 are related to neoplastic transformation and progression of the disease and these microRNAs could serve as a potential diagnostic and prognostic biomarkers in esophageal cancer., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4646922201567057.

Background. Sunitinib is a tyrosine kinase inhibitor used in the treatment of metastatic renal cell carcinoma. The main difficulty related to the treatment is the development of drug resistance followed by rapid progression of the disease. We analyzed tumor tissue of sunitinib treated patients in order to find miRNAs associated with therapeutic response. Methods. A total of 79 patients with metastatic renal cell carcinoma were included in our study. miRNA profiling in tumor tissue samples was performed by TaqMan Low Density Arrays and a group of selected miRNAs (miR-155, miR-374-5p, miR-324-3p, miR-484, miR-302c, and miR-888) was further validated by qRT-PCR. Normalized data were subjected to ROC and Kaplan-Meier analysis. Results. We reported decreased tissue levels of miR-155 and miR-484 as significantly associated with increased time to progression (miR-155: median TTP 5.8 versus 12.8 months, miR-484: median TTP 5.8 versus 8.9 months). Conclusion. miR-155 and miR-484 are potentially connected with sunitinib resistance and failure of the therapy. miR-155 is a known oncogene with direct influence on neovascularization. Biological role of miR-484 has to be clarified. Stratification of patients based on miRNA analysis would allow more personalized approach in therapy of metastatic renal cell carcinoma.

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., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

MicroRNAs (miRNAs) are short, 18-25-nucleotide long, non-coding single-stranded RNAs, which are capable to regulate gene expression on post-transcriptional level through binding to their target protein-encoding mRNAs. miRNAs regulate individual components of multiple oncogenic pathways. One of them is epidermal growth factor receptor (EGFR) signalling pathway that regulates cell proliferation, differentiation, migration, angiogenesis and apoptosis. All these processes are deregulated in colorectal cancer (CRC). Moreover, EGFR has been validated as the therapeutic target in CRC, and monoclonal antibodies cetuximab and panitumumab are used in the therapy of patients with metastatic CRC. Because of the extensive involvement of miRNAs in the regulation of EGFR signalling, it seems they could also serve as promising predictive biomarkers to anti-EGFR therapy. In this review, we summarize current knowledge about miRNAs targeting EGFR signalling pathway, their functioning in CRC pathogenesis and potential usage as biomarkers.

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.

Sporadic colorectal cancer (CRC) is a typical multifactorial disease. Isothiocyanates (ITC) have been recently shown to inhibit development of CRC in many experimental models. MicroRNAs (miRNAs) are short noncoding RNAs that posttranscriptionally regulate gene expression through binding to 3' untranslated regions (3'UTR) of target mRNAs. MiRNAs are regulated by natural agents, ITCs included. In our study, using global expression profiling based on TaqMan Low-Density Arrays, we identified 3 common miRNAs (miR-155, miR-23b, miR-27b) regulated by ITCs (sulforaphane, iberin) in colonic epithelial cell lines NCM460 and NCM356. In silico predictions allowed us to find 9 relevant single nucleotide polymorphisms (SNPs) localized within the 3'UTRs of genes (AGTR1, TNFAIP2, PRKCB, HSPA9, RABGAP1, DICER1, ADAM19, VWA5A, and SIRT5) targeted by these ITC-related miRNAs. Finally, we observed that homozygous CC genotype of DICER1, rs1057035, was significantly associated with decreased risk of CRC (odds ratio = 0.49; 95% confidence interval: 0.25-0.95, P = 0.036) when compared to TT homozygote genotype; also, the C allele tended to have a protective effect (P = 0.072). This study showed that miRNAs could be involved in chemoprotective effects of natural agents; their function alteration through SNPs in their binding sites and flanking regions presents a new class of CRC risk factors.

Background: Rectal 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., Methods: We 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., Results: Expression 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., Conclusions: Our 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.

Purpose: MicroRNA-21 (miR-21) is one of the miRNAs that are frequently and highly overexpressed in tumor tissue of colorectal cancer (CRC) patients; however, only a little is known about its functional role in CRC., Methods: We examined the expression level of miR-21 in 44 paired samples of tumoral and non-tumoral colon tissues diagnosed for CRC using TaqMan real-time PCR method. Furthermore, we used miR-21 inhibitor (anti-miR-21) to transient knockdown of miR-21 in DLD-1 colon cancer cells and examined the effects of miR-21 silencing on viability, apoptosis, chemosensitivity, cell cycle, and migration of DLD1 cells., Results: The expression levels of miR-21 were significantly increased in CRC tumor tissue (P < 0.0001). Significant differences in miR-21 levels were observed also between CRC tissues of patients with CRC in different clinical stages: I vs. II (P = 0.033) and I vs. IV (P = 0.021). Kaplan-Meier analysis proved that the miR-21 expression levels are correlated to shorter overall survival of CRC patients (P = 0.0341). MiR-21 silencing in DLD1 cell line had no effect on the cell viability; however, when combined with chemotherapeutics (5-FU, L-OHP, and SN38), it contributed to the decrease of cell viability. Suppression of miR-21 decreased cell migration ability of DLD-1 cells by nearly 30 % (P = 0.016)., Conclusion: We have confirmed the overexpression of miR-21 in CRC samples and its correlation with advanced disease and shorter overall survival. These findings could be described in part by the fact that CRC cells with increased expression of miR-21 have higher migration ability.

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., (© 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

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., (Copyright © 2012 Wiley Periodicals, Inc.)

Aim: To investigate whether selected single nucleotide polymorphisms (SNPs) in miR-196a2, miR-27a and miR-146a genes are associated with sporadic colorectal cancer (CRC)., Methods: In order to investigate the effect of these SNPs in CRC, we performed a case-control study of 197 cases of sporadic CRC and 212 cancer-free controls originating from the Central-European Caucasian population using TaqMan Real-Time polymerase chain reaction and allelic discrimination analysis., Results: The genotype and allele frequencies of SNPs were compared between the cases and the controls. None of the performed analysis showed any statistically significant results., Conclusion: Our data suggest a lack of association between rs11614913, rs895819 and rs2910164 and colorectal cancer risk in the Central-European Caucasian population, a population with an extremely high incidence of sporadic colorectal cancer.

Background: Breast cancer is the most common malignancy with the highest incidence rates among women worldwide. Triple-negative breast cancer (TNBC) represents the major phenotype of basal-like molecular subtype of breast cancer, characterized by higher incidence in young women and a very poor prognosis. MicroRNAs (miRNAs) are small non-coding RNAs playing significant role in the pathogenesis of many cancers including breast cancer. Therefore, miRNAs are also potential prognostic and/or predictive biomarkers in triple-negative breast cancer patients., Methods: Thirty-nine TNBC patients with available formalin-fixed paraffin-embedded (FFPE) tissues were enrolled in the study. MiR-34a, miR-34b, and miR-34c were analyzed using qRT-PCR and correlated to clinico-pathological features of TNBC patients., Results: Expression levels of miR-34b significantly correlate with disease free survival (DFS) (p = 0.0020, log-rank test) and overall survival (OS) (p = 0.0008, log-rank test) of TNBC patients. No other significant associations between miR-34a, miR-34b, and miR-34c with available clinical pathological data were observed., Conclusions: MiR-34b expression negatively correlates with disease free survival and overall survival in TNBC patients. Thus, miR-34b may present a new promising prognostic biomarker in TNBC patients, but independent validations are necessary.

Background: There 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., Methods: In 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., Results: We 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., Conclusions: Circulating miRNAs in serum are promising biomarkers in RCC.

Background: Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the world. Therefore, there is a high demand for cost-effective and non-invasive biomarkers that would enable an early detection of asymptomatic and curable disease with high sensitivity and specificity., Objective: The main objective of this study was to investigate the potential of circulating miRNAs as biomarkers of CRC., Methods: Total RNA enriched for small RNAs was isolated from 100~sera of patients with CRC and 30 sera of healthy donors. The expression levels of miR-17-3p, miR-29a, miR-92a and miR-135b were determined using quantitative real-time PCR. The average expression levels of particular miRNAs were normalized to miR-16 levels and statistically evaluated., Results: Using Mann-Whitney U test, no significant differences were observed in miR-17-3p (P=0.18), miR-29a (P=0.14) and miR-92a (P=0.60) levels between sera of CRC patients and controls. The levels of miR-135b in serum were too low to be quantified accurately. Subsequently, we tried to correlate expression levels of analyzed miRNAs to clinical-pathological features of CRC patients. Only levels of mir-29a were correlated with the clinical stage (P=0.04). Expression levels of the other miRNAs were correlated neither with the clinical stage, nor with the grade., Conclusions: Interestingly, our results are contradictory to previous studies performed on the CRC patients from Chinese population, providing an evidence against usage of serum miR-17-3p, miR-29a, miR-92a and miR-135b as new biomarkers for early detection of CRC.

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression. Single nucleotide polymorphisms (SNPs) may occur in miRNA biogenesis pathway genes, primary miRNA, pre-miRNA or a mature miRNA sequence. Such polymorphisms may be functional with respect to biogenesis and actions of mature miRNA. Specific SNPs were identified in predicted miRNA target sites within 3' untranslated regions of mRNAs. These SNPs have a potential to affect the efficiency of miRNA binding to the target sites or can create or disrupt binding sites. Resulting gene dysregulation may involve changes in phenotype and may eventually prove critical for the susceptibility to cancer and its onset as well as for estimates of prognosis and therapy response. In this review, we provide a comprehensive list of potentially functional miRNA-related SNPs and summarize their importance as candidate cancer biomarkers., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Glioblastoma multiforme (GBM) is the most frequently occurring primary malignant brain tumor; patients with GBM often have a very poor prognosis and differing responses to treatment. Therefore, it is very important to find new biomarkers that can predict clinical outcomes and help in treatment decisions. MicroRNAs are small, non-coding RNAs that function as post-transcriptional regulators of gene expression and play a key role in the pathogenesis of GBM. In a group of 38 patients with primary GBM, we analyzed the expression of eight microRNAs (miR-21, miR-128a, miR-181c, miR-195, miR-196a, miR-196b, miR-221, and miR-222). In addition, we examined the methylation status of O-6-methylguanine-DNA methyltransferase (MGMT) promoter by high-resolution melting analysis, as this has been shown to be a predictive marker in GBM. MGMT methylation status correlated with progression-free survival (P = 0.0201; log-rank test) as well as with overall survival (P = 0.0054; log-rank test). MiR-195 (P = 0.0124; log-rank test) and miR-196b (P = 0.0492; log-rank test) positively correlated with overall survival. Evaluation of miR-181c in combination with miR-21 predicted time to progression within 6 months of diagnosis with 92% sensitivity and 81% specificity (P < 0.0001). Our data confirmed that the methylation status of MGMT but also miR-21, miR-181c, miR-195, and miR-196b to be associated with survival of GBM patients. Above all, we suggest that the combination of miR-181c and miR-21 could be a very sensitive and specific test to identify patients at high risk of early progression after surgery., (© 2011 Japanese Cancer Association.)

MicroRNAs (miRNAs) are non-protein-coding short single stranded RNAs in the size range 19-25 nucleotides that are associated with gene regulation at the transcriptional and translational level. Recent studies have proved that miRNAs play important roles in a large number of biological processes, including cellular differentiation, proliferation, apoptosis, etc. Changes in their expression were found in a variety of human cancers, including renal cell carcinoma pathogenesis. Specific miRNA alterations were associated with key pathogenetic mechanisms of renal cell carcinoma like hypoxia or epithelial-mesenchymal transition. In this review, we summarize the current knowledge of miRNA functions in renal cell carcinoma with an emphasis on miRNAs potential to serve as a powerful biomarker of disease and a novel therapeutic target in oncology., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: MicroRNAs are endogenously expressed regulatory noncoding RNAs. Previous studies have shown altered expression levels of several microRNAs in renal cell carcinoma., Methods: We examined the expression levels of selected microRNAs in 38 samples of conventional renal cell carcinoma (RCC) and 10 samples of non-tumoral renal parenchyma using TaqMan real-time PCR method., Results: The expression levels of miRNA-155 (p < 0.0001), miRNA-210 (p < 0.0001), miRNA-106a (p < 0.0001) and miRNA-106b (p < 0.0001) were significantly over-expressed in tumor tissue, whereas the expression of miRNA-141 (p < 0.0001) and miRNA-200c (p < 0.0001) were significantly decreased in RCC samples. There were no significant differences between expression levels of miRNA-182 and miRNA-200b in tumor samples and renal parenchyma. Our data suggest that expression levels of miRNA-106b are significantly lower in tumors of patients who developed metastasis (p = 0.030) and miR-106b is a potential predictive marker of early metastasis after nephrectomy in RCC patients (long-rank p = 0.032)., Conclusions: We have confirmed previous observations obtained by miRNA microarray analysis using standardized real-time PCR method. For the first time, we have identified a prognostic significance of miRNA-106b, which, after validation on a larger group of patients, maybe useful as a promising biomarker in patients with RCC.

MicroRNAs are endogenously expressed regulatory noncoding RNAs. Previous studies showed altered expression levels of several microRNAs in glioblastomas. In this study, we examined the expression levels of selected microRNAs in 22 primary glioblastomas and six specimens of adult brain tissue by real-time PCR method. In addition, we examined methylation status of MGMT promoter by methylation-specific real-time PCR, as this has been shown to be a predictive marker in glioblastomas. MGMT methylation status was not correlated with response to concomitant chemoradiotherapy with temozolomide (RT/TMZ). MiR-221 (p=0.016), miR-222 (p=0.038), miR-181b (p=0.036), miR-181c (p=0.043) and miR-128a (p=0.001) were significantly down-regulated in glioblastomas. The most significant change was observed for up-regulation in miR-21 expression in glioblastomas (p<0.001). MiR-181b and miR-181c were significantly down-regulated in patients who responded to RT/TMZ (p=0.016; p=0.047, respectively) in comparison to patients with progredient disease. Our data indicate for the first time that expression levels of miR-181b and miR-181c could serve as a predictive marker of response to RT/TMZ therapy in glioblastoma patients.

MicroRNAs (miRNAs) are small non-coding RNAs 18-25 nucleotides in length that downregulate gene expression during various crucial cell processes such as apoptosis, differentiation and development. Changes in the expression profiles of miRNAs have been observed in a variety of human tumors, including colorectal cancer (CRC). Functional studies indicate that miRNAs act as tumor suppressors and oncogenes. These findings significantly extend Vogelstein's model of CRC pathogenesis and have shown great potential for miRNAs as a novel class of therapeutic targets. Several investigations have also described the ability of miRNA expression profiles to predict prognosis and response to selected treatments in CRC patients, and support diagnosis of CRC among cancer of unknown primary site. miRNAs' occurrence has been repeatedly observed also in serum and plasma, and miRNAs as novel minimally invasive biomarkers have indicated reasonable sensitivity for CRC detection and compare favorably with the fecal occult blood test. In this review, we summarize the knowledge regarding miRNAs' functioning in CRC while emphasizing their significance in pathogenetic signaling pathways and their potential to serve as disease biomarkers and novel therapeutic targets.

Renal cell carcinoma accounts for approximately 3% of adult cancers and has the highest lethality of urological malignancies. Research focusing on carcinogenesis and development of renal cell carcinoma has led to the identification of the key signalling pathways and consequently targeted cancer therapy which improves time to progression or overall survival of renal cell carcinoma patients. Today, microarray technologies are some of the most efficient methods used in gene expression studies. Through one microarray experiment we can simultaneously determine the expression of thousands of genes, thus facilitating research of examined biological models. The most frequently used of the microarray technologies are DNA microarrays enabling global analysis of the mRNA (messenger RNA) expression, while recently, microarray platforms modified to detect short non-coding RNAs (microRNAs) have been employed (microRNA microarrays). MicroRNAs significantly affect the behaviour of tumour cells by post-transcriptional regulation of the gene expression. In the research into renal cell carcinoma, microarray technologies have been applied in more than twenty studies over the past five years. These papers describe the potential of microarrays to distinguish tumour tissue from normal renal parenchyma, to classify renal cell carcinomas according to histological subtypes, to identify expression profiles predicting metastasizing in primary renal tumours, and to determine the prognosis of particular renal cell carcinoma patients. The aim of this review is to summarize the results from microarray studies of renal cell carcinoma realized to date and to present their potential usage in diagnostic and therapeutic protocols.

There is increasing evidence that some microRNAs change their levels in reaction to xenobiotic challenge. The aim of this study was to test the possible involvement of micro-RNAs in response to standard anticancer treatment. Tumor biopsies from 35 patients with rectal cancer before therapy and parallel tumor biopsies from 31 patients two weeks after starting preoperative capecitabine chemoradiotherapy were taken. The expression levels of single miRNA species were measured using TaqMan Micro-RNA assays after reverse transcription from isolated total RNAs. Many micro-RNAs (miR10a, miR21, miR145, miR212, miR339, miR361) responded to chemoradiotherapy in individual tumor samples, but there was profound intertumoral variability. However, other two micro-RNAs miR125b, miR137 showed a significant increase in median expression levels after starting therapy in most samples. Moreover, our results for the first time show that higher induced levels of miR125b and miR137 are associated with worse response to the therapy.

MicroRNAs (miRNAs) are large class of non-coding RNAs that post-transcriptionally regulate gene expression. Their ability of translational repression applied for example on oncogenes or tumor-suppressor genes indicates involvement of miRNAs in multi-step carcinogenesis. Evidences of miRNAs linkage to biological processes like apoptosis, proliferation, differentiation and cell survival are rapidly accumulating. Approximately 50% of miRNAs are located at fragile sites of chromosomes or regions known to be amplified or deleted in human cancer. That is why, non-coding miRNAs seem to be another level of genetic information which regulation is altered or lost during neoplastic growth. Expression profiles of miRNAs are successfully used for molecular classification, more exact diagnosis and prognosis of human cancers and reached analogical analytical characteristics like studies based on DNA micro-arrays technology and profiling of coding transcripts. In this review we attempt to introduce basic knowledge of miRNAs biogenesis and biological functions and in particular summarise reports focused on miRNAs in oncology research area.

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., ((c) 2008 S. Karger AG, Basel)