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

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.

Proteomic analysis of the bone marrow microenvironment in extramedullary multiple myeloma patients.

Multiple myeloma (MM) is a heterogeneous hematological malignancy characterized by the uncontrolled clonal proliferation of bone marrow (BM) plasma cells. The poor prognosis of patients is associated with the presence of extramedullary disease (EMD). Previously, different mechanisms involved in the colonization of BM niches by MM cells and their escape during EMD have been described. Thus, we aimed to investigate the expression of selected cytokines in the BM plasma of MM patients as well as EMD patients to reveal novel molecules involved in EMD pathogenesis. Expression of 120 different cytokines was measured in BM plasma of 13 MM and 11 EMD patients using Proteome Profiler Antibody Arrays. The correlation between statistically significant cytokines and clinicopathological parameters of patients was determined using the Spearman correlation analysis. Finally, protein-protein interactions were analyzed, and GO and KEGG pathways enrichment analysis was performed. In total, 27 cytokines were found to be differently expressed between MM and EMD patients. After the Benjamini-Hochberg correction for multiple testing, the statistical significance of two cytokines downregulated in EMD (EGF, BDNF) and six cytokines upregulated in EMD (NAP-2, ADIPOQ, CRP, MIG, BAFF, and THBS1) was maintained. Correlation analysis proved a significant association between the expression of these molecules and selected clinical-pathological features of MM/EMD patients. Protein association network analysis revealed important protein-protein interactions between THBS1/EGF, MIG/NAP-2, THBS1/NAP-2, EGF/NAP-2, and ADIPOQ/CRP. Finally, identified cytokines were proved to be significantly involved in focal adhesion, PI3K/AKT, and MAPK signaling pathways, and regulation of cell development, localization, proliferation, migration, differentiation, immune system processes, and stress response. Obtained results confirm the key function of the BM microenvironment in the pathogenesis of MM and indicate the essential role of numerous cytokines in disease progression and EMD development. However, the exact mechanisms need to be further clarified.

Involvement of Small Non-Coding RNA and Cell Antigens in Pathogenesis of Extramedullary Multiple Myeloma.

Extramedullary multiple myeloma (EMD) is an aggressive disease; malignant plasma cells lose their dependence in the bone marrow microenvironment and migrate into tissues. EMD is a negative prognostic factor of survival. Using flow cytometry and next-generation sequencing, we aimed to identify antigens and microRNAs (miRNAs) involved in EMD pathogenesis. Flow cytometry analysis revealed significant differences in the level of clonal plasma cells between MM and EMD patients, while the expression of CD markers was comparable between these two groups. Further, miR-26a-5p and miR-30e-5p were found to be significantly down-regulated in EMD compared to MM. Based on the expression of miR-26a-5p, we were able to distinguish these two groups of patients with high sensitivity and specificity. In addition, the involvement of deregulated miRNAs in cell cycle regulation, ubiquitin-mediated proteolysis and signaling pathways associated with infections or neurological disorders was observed using GO and KEGG pathways enrichment analysis. Subsequently, a correlation between the expression of analyzed miRNAs and the levels of CD molecules was observed. Finally, clinicopathological characteristics as well as CD antigens associated with the prognosis of MM and EMD patients were identified. Altogether, we identified several molecules possibly involved in the transformation of MM into EMD.

Therapeutic potential of FLANC, a novel primate-specific long non-coding RNA in colorectal cancer.

OBJECTIVE: To investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target. DESIGN: FLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases. RESULTS: FLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis. CONCLUSIONS: Based on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients.

miR-181a-2* expression is different amongst carcinomas from the colorectal serrated route.

Serrated adenocarcinoma (SAC) and colorectal carcinomas showing histological and molecular features of high-level of microsatellite instability (hmMSI-H) are both end points of the serrated pathway of colorectal carcinogenesis. Despite common features (right-sided location, CpG island methylation phenotype and BRAF mutation) there are no studies comparing the microRNA (miRNA) expression profiles in SACs and hmMSI-H. The microtranscriptome from 12 SACs and 8 hmMSI-H were analysed using Affymetrix GeneChip miRNA 3.0 arrays and differentially enriched functions involving immune response were observed from this comparison. miR-181a-2* was found significantly more expressed in hmMSI-H than in SAC and higher expression of this miRNA in microsatellite unstable colorectal cancer were corroborated by Real-Time PCR in an extended series (61 SAC, 21 hmMSI-H). An analysis of genes possibly regulated by miR-181a-2* was carried out and, amongst these, an inverse correlation of NAMPT with miR-181a-2* expression was observed, whereas, for TRAF1 and SALL1, additional regulation mechanisms involving CpG island methylation were observed. miR-181a-2* is associated with particular histological and molecular features of colorectal carcinomas within the serrated pathological pathway and might play a role in the immune responses of microsatellite instability carcinomas.

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.

Serum-based microRNA signatures in early diagnosis and prognosis prediction of colon cancer.

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.

Prediction of response to anti-EGFR antibody-based therapies by multigene sequencing in colorectal cancer patients.

BACKGROUND: The anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (moAbs) cetuximab or panitumumab are administered to colorectal cancer (CRC) patients who harbor wild-type RAS proto-oncogenes. However, a percentage of patients do not respond to this treatment. In addition to mutations in the RAS genes, mutations in other genes, such as BRAF, PI3KCA, or PTEN, could be involved in the resistance to anti-EGFR moAb therapy. METHODS: In order to develop a comprehensive approach for the detection of mutations and to eventually identify other genes responsible for resistance to anti-EGFR moAbs, we investigated a panel of 21 genes by parallel sequencing on the Ion Torrent Personal Genome Machine platform. We sequenced 65 CRCs that were treated with cetuximab or panitumumab. Among these, 37 samples were responsive and 28 were resistant. RESULTS: We confirmed that mutations in EGFR-pathway genes (KRAS, NRAS, BRAF, PI3KCA) were relevant for conferring resistance to therapy and could predict response (p = 0.001). After exclusion of KRAS, NRAS, BRAF and PI3KCA combined mutations could still significantly associate to resistant phenotype (p = 0.045, by Fisher exact test). In addition, mutations in FBXW7 and SMAD4 were prevalent in cases that were non-responsive to anti-EGFR moAb. After we combined the mutations of all genes (excluding KRAS), the ability to predict response to therapy improved significantly (p = 0.002, by Fisher exact test). CONCLUSIONS: The combination of mutations at KRAS and at the five gene panel demonstrates the usefulness and feasibility of multigene sequencing to assess response to anti-EGFR moAbs. The application of parallel sequencing technology in clinical practice, in addition to its innate ability to simultaneously examine the genetic status of several cancer genes, proved to be more accurate and sensitive than the presently in use traditional approaches.

Investigation of long non-coding RNAs in extracellular vesicles from low-volume blood serum specimens of colorectal cancer patients.

Colorectal cancer (CRC) is the second most prevalent cancer type worldwide, which highlights the urgent need for non-invasive biomarkers for its early detection and improved prognosis. We aimed to investigate the patterns of long non-coding RNAs (lncRNAs) in small extracellular vesicles (sEVs) collected from low-volume blood serum specimens of CRC patients, focusing on their potential as diagnostic biomarkers. Our research comprised two phases: an initial exploratory phase involving RNA sequencing of sEVs from 76 CRC patients and 29 healthy controls, and a subsequent validation phase with a larger cohort of 159 CRC patients and 138 healthy controls. Techniques such as dynamic light scattering, transmission electron microscopy, and Western blotting were utilized for sEV characterization. Optimized protocol for sEV purification, RNA isolation and preamplification was applied to successfully sequence the RNA content of sEVs and validate the results by RT-qPCR. We successfully isolated sEVs from blood serum and prepared sequencing libraries from a low amount of RNA. High-throughput sequencing identified differential levels of 460 transcripts between CRC patients and healthy controls, including mRNAs, lncRNAs, and pseudogenes, with approximately 20% being lncRNAs, highlighting several tumor-specific lncRNAs that have not been associated with CRC development and progression. The validation phase confirmed the upregulation of three lncRNAs (NALT1, AL096828, and LINC01637) in blood serum of CRC patients. This study not only identified lncRNA profiles in a population of sEVs from low-volume blood serum specimens of CRC patients but also highlights the value of innovative techniques in biomolecular research, particularly for the detection and analysis of low-abundance biomolecules in clinical samples. The identification of specific lncRNAs associated with CRC provides a foundation for future research into their functional roles in cancer development and potential clinical applications.

Identification and functional screening of microRNAs highly deregulated in colorectal cancer.

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

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.

Clinical correlations of miR-21 expression in colorectal cancer patients and effects of its inhibition on DLD1 colon cancer cells.

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.

Epigenetic Regulation of MicroRNA Clusters and Families during Tumor Development.

MicroRNAs are small non-coding single-stranded RNA molecules regulating gene expression on a post-transcriptional level based on the seed sequence similarity. They are frequently clustered; thus, they are either simultaneously transcribed into a single polycistronic transcript or they may be transcribed independently. Importantly, microRNA families that contain the same seed region and thus target related signaling proteins, may be localized in one or more clusters, which are in a close relationship. MicroRNAs are involved in basic physiological processes, and their deregulation is associated with the origin of various pathologies, including solid tumors or hematologic malignancies. Recently, the interplay between the expression of microRNA clusters and families and epigenetic machinery was described, indicating aberrant DNA methylation or histone modifications as major mechanisms responsible for microRNA deregulation during cancerogenesis. In this review, the most studied microRNA clusters and families affected by hyper- or hypomethylation as well as by histone modifications are presented with the focus on particular mechanisms. Finally, the diagnostic and prognostic potential of microRNA clusters and families is discussed together with technologies currently used for epigenetic-based cancer therapies.

LncRNAs LY86-AS1 and VIM-AS1 Distinguish Plasma Cell Leukemia Patients from Multiple Myeloma Patients.

Long non-coding RNAs (lncRNAs) are functional RNAs longer than 200 nucleotides. Due to modern genomic techniques, the involvement of lncRNAs in tumorigenesis has been revealed; however, information concerning lncRNA interplay in multiple myeloma (MM) and plasma cell leukemia (PCL) is virtually absent. Herein, we aimed to identify the lncRNAs involved in MM to PCL progression. We investigated representative datasets of MM and PCL patients using next-generation sequencing. In total, 13 deregulated lncRNAs (p < 0.00025) were identified; four of them were chosen for further validation in an independent set of MM and PCL patients by RT-qPCR. The obtained results proved the significant downregulation of lymphocyte antigen antisense RNA 1 (LY86-AS1) and VIM antisense RNA 1 (VIM-AS1) in PCL compared to MM. Importantly, these two lncRNAs could be involved in the progression of MM into PCL; thus, they could serve as promising novel biomarkers of MM progression.

microRNA-342, microRNA-191 and microRNA-510 are differentially expressed in T regulatory cells of type 1 diabetic patients.

Regulatory T cells (Tregs) are critical regulators of autoimmune diseases, including type 1 diabetes mellitus. It is hypothesised that Tregs' function can be influenced by changes in the expression of specific microRNAs (miRNAs). Thus, we performed miRNAs profiling in a population of Tregs separated from peripheral blood of five type 1 diabetic patients and six healthy donors. For more detailed molecular characterisation of Tregs, we additionally compared miRNAs expression profiles of Tregs and conventional T cells. Tregs were isolated according to CD3+, CD4+, CD25(hi)+ and CD127- by flow cytometry, and miRNA expression profiling was performed using TaqMan Array Human MicroRNA Panel-1 (384-well low density array). In Tregs of diabetic patients we found significantly increased expression of miRNA-510 (p=0.05) and decreased expression of both miRNA-342 (p<0.0001) and miRNA-191 (p=0.0079). When comparing Tregs and T cells, we revealed that Tregs had significant higher expression of miRNA-146a and lower expression of eight specific miRNAs (20b, 31, 99a, 100, 125b, 151, 335, and 365). To our knowledge, this is the first study demonstrating changes in miRNA expression profiles occurring in Tregs of T1D patients and a miRNAs signature of adult Tregs.

[Role of microRNAs in the immune system]. / Role mikroRNA v imunitním systému.

MicroRNAs are short non-coding RNAs involved in post-trascriptional regulation of gene expression. They can be found in various organisms including animals, plants and viruses and they play a key role in diverse biological processes, such as embryogenesis, differentiation and proliferation of cells, production of cytokines or apoptosis. Since the initial observation, more than 700 microRNAs have been identified in mammalian cells and up to one third of all protein-encoding genes is estimated to be regulated by these small molecules. In order to biological functions of these mRNA targets, it is apparent today, that altered microRNA expression can contribute not only to carcinogenesis but also to serious failure of the immune system. The aim of this review is to summarize the latest findings about the biogenesis and function of microRNAs and to refer on their role in the regulation of ontogenesis of immune system, haematopoiesis, immune responses and viral infections. Possible contribution of microRNAs to the therapy of viral infections is discussed.

MiR-215-5p Reduces Liver Metastasis in an Experimental Model of Colorectal Cancer through Regulation of ECM-Receptor Interactions and Focal Adhesion.

Background: Growing evidence suggests that miR-215-5p is a tumor suppressor in colorectal cancer (CRC); however, its role in metastasis remains unclear. This study evaluates the effects of miR-215 overexpression on the metastatic potential of CRC. Methods: CRC cell lines were stably transfected with miR-215-5p and used for in vitro and in vivo functional analyses. Next-generation sequencing and RT-qPCR were performed to study changes on the mRNA level. Results: Overexpression of miR-215-5p significantly reduced the clonogenic potential, migration, and invasiveness of CRC cells in vitro and tumor weight and volume, and liver metastasis in vivo. Transcriptome analysis revealed mRNAs regulated by miR-215-5p and RT-qPCR confirmed results for seven selected genes. Significantly elevated levels of CTNNBIP1 were also observed in patients' primary tumors and liver metastases compared to adjacent tissues, indicating its direct regulation by miR-215-5p. Gene Ontology and KEGG pathway analysis identified cellular processes and pathways associated with miR-215-5p deregulation. Conclusions: MiR-215-5p suppresses the metastatic potential of CRC cells through the regulation of divergent molecular pathways, including extracellular-matrix-receptor interaction and focal adhesion. Although the specific targets of miR-215-5p contributing to the formation of distant metastases must be further elucidated, this miRNA could serve as a promising target for CRC patients' future therapeutic strategies.

MicroRNA-215: From biology to theranostic applications.

MicroRNA-215 (miR-215) is one of the extensively studied microRNAs (miRNAs) in human diseases, especially in different types of cancer, where it plays various roles in the initiation and progression of tumors. There is also a high conservation of miR-215 among wide range of different species indicating that this miRNA may have vital functions that are maintained during evolution. During the last ten years, significant efforts were dedicated to uncover molecular mechanisms of miR-215 regulation and cellular functioning. In addition, miR-215 was repeatedly identified to have the causal roles in pathology of various diseases, where it may serve as a promising diagnostic, prognostic or predictive biomarker and as a therapeutic target. Here, we overview more than 150 reports focused on miR-215 to allow the synthesis of knowledge on its transcriptional and post-transcriptional regulation, and functioning in essential biological processes like cell and tissue development, cell survival, cell cycle and proliferation, cell migration and invasion, cellular microenvironment communication, and metabolism. Further, we summarized the findings on miR-215 roles in pathology of nonmalignant diseases and various types of cancer with special focus on miR-215 as a promising molecule for future development of theranostic miRNA-based approaches. Although it is difficult to evaluate the full theranostic potential of miR-215, it is probable that during the following years, an increasing number of theranostic miRNA modalities that overcome the current technological obstacles will appear, and enable the translation of miR-215 knowledge into the clinic.

Long Non-Coding RNAs in Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematooncological disease of malignant plasma cells in the bone marrow. While new treatment brought unprecedented increase of survival of patients, MM pathogenesis is yet to be clarified. Increasing evidence of expression of long non-coding RNA molecules (lncRNA) linked to development and progression of many tumors suggested their important role in tumorigenesis. To date, over 15,000 lncRNA molecules characterized by diversity of function and specificity of cell distribution were identified in the human genome. Due to their involvement in proliferation, apoptosis, metabolism, and differentiation, they have a key role in the biological processes and pathogenesis of many diseases, including MM. This review summarizes current knowledge of non-coding RNAs (ncRNA), especially lncRNAs, and their role in MM pathogenesis. Undeniable involvement of lncRNAs in MM development suggests their potential as biomarkers.

Pre-operative Plasma miR-21-5p Is a Sensitive Biomarker and Independent Prognostic Factor in Patients with Pancreatic Ductal Adenocarcinoma Undergoing Surgical Resection.

Blood plasma microRNAs (miRNAs) are emerging as a clinically useful tool for non-invasive detection and prognosis estimation in various cancer types including pancreatic ductal adenocarcinoma (PDAC). The aim of the present study was to provide an independent validation of circulating miRNAs identified in previous studies as diagnostic and/or prognostic biomarkers in PDAC. Based on the literature search, 6 miRNAs were chosen as candidates for independent validation; miR-21-5p, miR-375, miR-155, miR-17-5p, miR-126-5p and miR-1290. Validation of these miRNAs was performed in a cohort of 25 patients with PDAC undergoing surgical resection and 24 healthy donors. Plasma levels of miRNAs were determined using quantitative real-time PCR. We confirmed significantly higher levels of all tested miRNA in blood plasma of PDAC patients in comparison to healthy controls with miR-21-5p showing the highest analytical performance (p<0.001; AUC>0.99). Increased levels of miR-21-5p (p=0.045) and miR-375 (p=0.013) were significantly associated with overall survival. Multivariate analysis demonstrated that miR-21-5p is a significant unfavorable prognostic factor independent on other clinical variables including adjuvant chemotherapy (hazard ratio 2.95; 95% CI 1.06-8.18; p=0.038). Our preliminary data indicate promising diagnostic and prognostic utility of plasma miR-21-5p in PDAC patients.