Jiri Sana

Novel classes of non-coding RNAs and cancer

For the many years, the central dogma of molecular biology has been that RNA functions mainly as an informational intermediate between a DNA sequence and its encoded protein. But one of the great surprises of modern biology was the discovery that protein-coding genes represent less than 2% of the total genome sequence, and subsequently the fact that at least 90% of the human genome is actively transcribed. Thus, the human transcriptome was found to be more complex than a collection of protein-coding genes and their splice variants. Although initially argued to be spurious transcriptional noise or accumulated evolutionary debris arising from the early assembly of genes and/or the insertion of mobile genetic elements, recent evidence suggests that the non-coding RNAs (ncRNAs) may play major biological roles in cellular development, physiology and pathologies. NcRNAs could be grouped into two major classes based on the transcript size; small ncRNAs and long ncRNAs. Each of these classes can be further divided, whereas novel subclasses are still being discovered and characterized. Although, in the last years, small ncRNAs called microRNAs were studied most frequently with more than ten thousand hits at PubMed database, recently, evidence has begun to accumulate describing the molecular mechanisms by which a wide range of novel RNA species function, providing insight into their functional roles in cellular biology and in human disease. In this review, we summarize newly discovered classes of ncRNAs, and highlight their functioning in cancer biology and potential usage as biomarkers or therapeutic targets.

MiR-195, miR-196b, miR-181c, miR-21 expression levels and O-6-methylguanine-DNA methyltransferase methylation status are associated with clinical outcome in glioblastoma patients

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. (Cancer Sci 2011; 102: 2186–2190)

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.

MicroRNAs and glioblastoma: roles in core signalling pathways and potential clinical implications.

•  Introduction •  miRNAs signatures of glioblastoma tissue and cell lines •  Involvement of miRNAs in core glioblastoma signalling pathways ‐  EGFR and PI3K/AKT signalling pathways ‐  p53, TGF‐β and apoptotic signalling pathways ‐  IFN‐α/IFN‐β signalling pathways ‐  Notch signalling pathway ‐  NF‐κB signalling pathway •  Single nucleotide polymorphisms and miRNAs: risk factors for glioblastoma •  miRNAs in glioblastoma prognosis and prediction of therapeutic response •  miRNAs involved in drug resistance of glioblastoma •  miRNAs as potential therapeutic targets •  Conclusions

The role of microRNAs in mitochondria in cancer

Reprogramming of the energy metabolism in cancer cell presents progressive field of cancer research. This feature is associated mainly with the mitochondria, which is major energy source of eukaryotic cells. These organelles are essential to the cell homeostasis maintenance, play an important role in intrinsic apoptotic pathway and their dysfunction is associated with multiple diseases including cancer. Recently, it was described that microRNAs (miRNAs) regulate important signaling pathways in mitochondria and many of these miRNAs are deregulated in various cancers. Here we summarize current knowledge about miRNAs involved in mitochondrial functioning with focus on cellular energy metabolism, apoptosis and mitophagy.

MiR-34b is associated with clinical outcome in triple-negative breast cancer patients

BackgroundBreast 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.MethodsThirty-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.ResultsExpression 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.ConclusionsMiR-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.

MicroRNAs involved in chemo- and radioresistance of high-grade gliomas

High-grade gliomas (HGGs) are malignant primary brain tumors of glial cell origin. Despite optimal course of treatment, including maximal surgical resection followed by adjuvant chemo- and/or radiotherapy, the prognosis still remains poor. The main reason is the commonly occurring chemo- and radioresistance of these tumors. In recent years, several signaling pathways, especially PI3K/AKT and ATM/CHK2/p53, have been linked to the resistance of gliomas. Moreover, additional studies have shown that these pathways are significantly regulated by microRNAs (miRNAs), short endogenous RNA molecules that modulate gene expression and control many biological processes including apoptosis, proliferation, cell cycle, invasivity, and angiogenesis. MiRNAs are not only highly deregulated in gliomas, their expression signatures have also been shown to predict prognosis and therapy response. Therefore, they present promising biomarkers and therapeutic targets that might overcome the resistance to treatment and improve prognosis of glioma patients. In this review, we summarize the current knowledge of the functional role of miRNAs in gliomas resistance to chemo- and radiotherapy.

Prevalence of Propionibacterium acnes in Intervertebral Discs of Patients Undergoing Lumbar Microdiscectomy: A Prospective Cross-Sectional Study

Background The relationship between intervertebral disc degeneration and chronic infection by Propionibacterium acnes is controversial with contradictory evidence available in the literature. Previous studies investigating these relationships were under-powered and fraught with methodical differences; moreover, they have not taken into consideration P. acnes’ ability to form biofilms or attempted to quantitate the bioburden with regard to determining bacterial counts/genome equivalents as criteria to differentiate true infection from contamination. The aim of this prospective cross-sectional study was to determine the prevalence of P. acnes in patients undergoing lumbar disc microdiscectomy. Methods and Findings The sample consisted of 290 adult patients undergoing lumbar microdiscectomy for symptomatic lumbar disc herniation. An intraoperative biopsy and pre-operative clinical data were taken in all cases. One biopsy fragment was homogenized and used for quantitative anaerobic culture and a second was frozen and used for real-time PCR-based quantification of P. acnes genomes. P. acnes was identified in 115 cases (40%), coagulase-negative staphylococci in 31 cases (11%) and alpha-hemolytic streptococci in 8 cases (3%). P. acnes counts ranged from 100 to 9000 CFU/ml with a median of 400 CFU/ml. The prevalence of intervertebral discs with abundant P. acnes (≥ 1x103 CFU/ml) was 11% (39 cases). There was significant correlation between the bacterial counts obtained by culture and the number of P. acnes genomes detected by real-time PCR (r = 0.4363, p<0.0001). Conclusions In a large series of patients, the prevalence of discs with abundant P. acnes was 11%. We believe, disc tissue homogenization releases P. acnes from the biofilm so that they can then potentially be cultured, reducing the rate of false-negative cultures. Further, quantification study revealing significant bioburden based on both culture and real-time PCR minimize the likelihood that observed findings are due to contamination and supports the hypothesis P. acnes acts as a pathogen in these cases of degenerative disc disease.

CD133 Expression and Identification of CD133/nestin Positive Cells in Rhabdomyosarcomas and Rhabdomyosarcoma Cell Lines

Background: Co-expression of CD133, cell surface glycoprotein, and nestin, an intermediate filament protein, was determined to be a marker of neural stem cells and of cancer stem cells in neurogenic tumors. Methods: We examined the expression of CD133 and nestin in ten tumor tissue samples taken from patients with rhabdomyosarcomas and in five rhabdomyosarcoma cell lines. Immunohistochemistry and immunofluorescence were used to examine FFPE tumor tissue samples. Cell lines were analyzed by immunofluorescence, immunoblotting, flow cytometry, and RT-PCR. Functional assays (clonogenic in vitro assay and tumorigenic in vivo assay) were also performed using these cell lines. Results: CD133 and nestin were detected in all 10 tumor tissue samples and in all 5 cell lines; however, the frequency of CD133+, Nes+, and CD133+/Nes+ cells, as well as the intensity of fluorescence varied in individual samples or cell lines. The expression of CD133 and nestin was subsequently confirmed in all cell lines by immunoblotting. Furthermore, we observed an increasing expression of CD133 in relation to the cultivation. All cell lines were positive for Oct3/4 and nucleostemin; NSTS-11 cells were also able to form xenograft tumors in mice. Conclusion: Our results represent the first evidence of CD133 expression in rhabdomyosarcoma tissue and in rhabdomyosarcoma cell lines. In addition, the co-expression of CD133 and nestin as well as results of the functional assays suggest a possible presence of cancer cells with a stem-like phenotype in these tumors.

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.