Epithelial-mesenchymal transition-associated microRNA/mRNA signature is linked to metastasis and prognosis in clear-cell renal cell carcinoma

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

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 (>= 1x10(3) 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

Propionibacterium acnes biofilm is present in intervertebral discs of patients undergoing microdiscectomy

Background In previous studies, Propionibacterium acnes was cultured from intervertebral disc tissue of similar to 25% of patients undergoing microdiscectomy, suggesting a possible link between chronic bacterial infection and disc degeneration. However, given the prominence of P. acnes as a skin commensal, such analyses often struggled to exclude the alternate possibility that these organisms represent perioperative microbiologic contamination. This investigation seeks to validate P. acnes prevalence in resected disc cultures, while providing microscopic evidence of P. acnes biofilm in the intervertebral discs. Methods Specimens from 368 patients undergoing microdiscectomy for disc herniation were divided into several fragments, one being homogenized, subjected to quantitative anaerobic culture, and assessed for bacterial growth, and a second fragment frozen for additional analyses. Colonies were identified by MALDI-TOF mass spectrometry and P. acnes phylotyping was conducted by multiplex PCR. For a sub-set of specimens, bacteria localization within the disc was assessed by microscopy using confocal laser scanning and FISH. Results Bacteria were cultured from 162 discs (44%), including 119 cases (32.3%) with P. acnes. In 89 cases, P. acnes was cultured exclusively;in 30 cases, it was isolated in combination with other bacteria (primarily coagulase-negative Staphylococcus spp.) Among positive specimens, the median P. acnes bacterial burden was 350 CFU/g (12 - similar to 20,000 CFU/g). Thirtyeight P. acnes isolates were subjected to molecular sub-typing, identifying 4 of 6 defined phylogroups: IA1, IB, IC, and II. Eight culture-positive specimens were evaluated by fluorescence microscopy and revealed P. acnes in situ. Notably, these bacteria demonstrated a biofilm distribution within the disc matrix. P. acnes bacteria were more prevalent in males than females (39% vs. 23%, p = 0.0013). Conclusions This study confirms that P. acnes is prevalent in herniated disc tissue. Moreover, it provides the first visual evidence of P. acnes biofilms within such specimens, consistent with infection rather than microbiologic contamination

Translational Potential of MicroRNAs for Preoperative Staging and Prediction of Chemoradiotherapy Response in Rectal Cancer

Colorectal cancer is the third most common cancer and the second cause of cancer-related deaths. Rectal cancer presents roughly one-third of all colorectal cancer cases and differs from it on both anatomical and molecular levels. While standard treatment of colon cancer patients is radical surgery, rectal cancer is usually treated with pre-operative chemoradiotherapy followed by total mesorectal excision, which requires precise estimation of TNM staging. Unfortunately, stage evaluation is based solely on imaging modalities, and they often do not correlate with postoperative pathological findings. Moreover, approximately half of rectal cancer patients do not respond to such pre-operative therapy, so they are exposed to its toxic effects without any clinical benefit. Thus, biomarkers that could precisely predict pre-operative TNM staging, and especially response to therapy, would significantly advance rectal cancer treatment&mdash;but till now, no such biomarker has been identified. In cancer research, microRNAs are emerging biomarkers due to their connection with carcinogenesis and exceptional stability. Circulating miRNAs are promising non-invasive biomarkers that could allow monitoring of a patient throughout the whole therapeutic process. This mini-review aims to summarize the current knowledge on miRNAs and circulating miRNAs involved in the prediction of response to treatment and pre-operative staging in rectal cancer patients

Strategies for labelling of exogenous and endogenous extracellular vesicles and their application for in vitro and in vivo functional studies

Abstract This review presents a comprehensive overview of labelling strategies for endogenous and exogenous extracellular vesicles, that can be utilised both in vitro and in vivo. It covers a broad spectrum of approaches, including fluorescent and bioluminescent labelling, and provides an analysis of their applications, strengths, and limitations. Furthermore, this article presents techniques that use radioactive tracers and contrast agents with the ability to track EVs both spatially and temporally. Emphasis is also placed on endogenous labelling mechanisms, represented by Cre-lox and CRISPR-Cas systems, which are powerful and flexible tools for real-time EV monitoring or tracking their fate in target cells. By summarizing the latest developments across these diverse labelling techniques, this review provides researchers with a reference to select the most appropriate labelling method for their EV based research

Cerebrospinal Fluid MicroRNA Signatures as Diagnostic Biomarkers in Brain Tumors

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

MicroRNA isolation and quantification in cerebrospinal fluid: A comparative methodical study.

Associated with the pathogenesis of many cancers, including brain tumors, microRNAs (miRNAs) present promising diagnostic biomarkers. These molecules have been also studied in cerebrospinal fluid (CSF), showing great potential as a diagnostic tool in patients with brain tumors. Even though there are some biological and technological factors that could affect the results and their biological and clinical interpretation, miRNA analysis in CSF is not fully standardized. This study aims to compare several RNA extraction and miRNA quantification approaches, including high-throughput technologies and individual miRNA detection methods, thereby contributing to the optimization and standardization of quantification of extracellular miRNAs in CSF. Such knowledge is essential for the potential use of miRNAs as diagnostic biomarkers in brain tumors

MicroRNA Biogenesis Pathway Genes Are Deregulated in Colorectal Cancer

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