Marek Mráz

MicroRNA isolation and stability in stored RNA samples

MicroRNAs (miRNAs) are small RNA molecules, which act as post-transcriptional regulators of a gene expression, with important functions within the cell physiology. Whilst many authors have focused on the study of miRNA expression in physiological and pathological processes, various technical variables related to miRNA isolation have simultaneously emerged and the stability of the stored miRNA samples has been questioned. A robust method for RNA isolation is essential for reproducible results and miRNAs instability in the stored samples would make for an alarming situation for most expression studies. Here these issues are discussed and we investigate the stability of miRNAs isolated from clinical samples of B lymphocytes (chronic lymphocytic leukemia) by the most commonly utilized method based on a Trizol/TRI-Reagent solution (RNAs stored at -80 degrees C). To assess the stability of miRNAs, a Real Time-PCR analysis was performed for a panel of 29 miRNAs from a freshly isolated RNA sample and after 14 days storage at -80 degrees C. Furthermore, a Real Time-PCR analysis was repeatedly performed for a stored RNA sample over a period of approximately 10 months. We observed high stability of isolated miRNAs and respective cDNAs. The reproducibility and efficiency of the Trizol/TRI-Reagent isolation method was also tested and compared to the mirVana Isolation kit (Ambion) and RNeasy kit (Qiagen). In conclusion, Trizol/TRI-Reagent based isolation is a robust reproducible method, and obtained miRNA samples do not show any tendency to degradation when properly stored and handled.

miR-34a, miR-29c and miR-17-5p are downregulated in CLL patients with TP53 abnormalities.

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MicroRNAs in chronic lymphocytic leukemia pathogenesis and disease subtypes

MicroRNAs (miRNAs) are short, non-coding RNAs, which function as evolutionary conserved regulators of a gene expression. They have essential roles in development, cell differentiation, proliferation, apoptosis and chromosome structure. MiRNAs constitute about 3–5% of predicted genes in the human genome (i.e. about 1000); and 20–30% of the protein-coding genes are estimated to be regulated by the miRNAs. The primary evidence that miRNAs possibly act as a novel class of oncogenes/tumor-suppressors comes from the discovery of the miR-15a and miR-16-1 in 13q14 region deleted in chronic lymphocytic leukemia (CLL). Moreover, miRNA signatures have been used to classify tumor types. There have recently been several reports on the miRNAs role in CLL pathogenesis and disease subtypes (according to IgVH mutation status). In this report, we will review the published observations and present our miRNA profiling data in aggressive CLL with TP53 abnormalities (deletion and/or mutation of p53 gene). We have identified a deregulated miRNA expression pattern (down regulation of miR-34a, miR-29 and miR-17-5p) in these samples, compared to cells with wild-type TP53. It has previously been shown that miR-34a is directly regulated by p53 and targets BCL-2, miR-29c regulates the MCL-1 and TCL-1 proto-oncogenes and the miR-17-5p targets important cell cycle regulatory molecules. Consequently, these three miRNAs could potentially play important roles in the pathogenesis of aggressive CLL.

MYB transcriptionally regulates the miR-155 host gene in chronic lymphocytic leukemia

Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of miR-155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.

MicroRNAs in B-cell lymphomas: how a complex biology gets more complex

MicroRNAs (miRNAs) represent important regulators of gene expression besides transcriptional control. miRNA regulation can be involved in the cell developmental fate decisions, but can also have more subtle roles in buffering stochastic fluctuations in gene expression. They participate in pathways fundamental to B-cell development like B-cell receptor (BCR) signalling, B-cell migration/adhesion, cell–cell interactions in immune niches, and the production and class-switching of immunoglobulins. miRNAs influence B-cell maturation, generation of pre-, marginal zone, follicular, B1, plasma and memory B cells. In this review, we discuss miRNAs with essential functions in malignant B-cell development (such as miR-150, miR-155, miR-21, miR-34a, miR-17-92 and miR-15-16). We also put these miRNAs in the context of normal B-cell differentiation, as this is intimately connected to neoplastic B-cell development. We review miRNAs’ role in the most common B-cell malignancies, including chronic lymphocytic leukaemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL). We focus on miR-contribution to the regulation of important signalling pathways (such as NF-κB, PI3K/AKT and TGF-β), BCR signalling and its modulators (such as PTEN, SHIP-1, ZAP-70, GAB1 and BTK), anti- and pro-apoptotic proteins (such as BCL2, MCL1, TCL1, BIM, p53 and SIRT1) and transcription factors (such as MYC, MYB, PU.1, FOXP1 and BCL6). We also discuss the association of miRNAs’ expression levels with the patients’ survival and response to therapy, summarizing their potential use as predictive and prognostic markers. Importantly, the targeting of miRNAs (like use of anti-miR-155 or miR-34a mimic) could provide a novel therapeutic approach as evidenced by tumour regression in xenograft mouse models and initial promising data from clinical trials.

MicroRNAs Regulate p21Waf1/Cip1 Protein Expression and the DNA Damage Response in Human Embryonic Stem Cells

Studies of human embryonic stem cells (hESCs) commonly describe the nonfunctional p53‐p21 axis of the G1/S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs, but p21 protein is not detectable. In this article, we provide evidence that expression of p21 protein is directly regulated by the microRNA (miRNA) pathway under standard culture conditions and after DNA damage. The DDR in hESCs leads to upregulation of tens of miRNAs, including hESC‐specific miRNAs such as those of the miR‐302 family, miR‐371‐372 family, or C19MC miRNA cluster. Most importantly, we show that the hESC‐enriched miRNA family miR‐302 (miR‐302a, miR‐302b, miR‐302c, and miR‐302d) directly contributes to regulation of p21 expression in hESCs and, thus, demonstrate a novel function for miR‐302s in hESCS. The described mechanism elucidates the role of miRNAs in regulation of important molecular pathway governing the G1/S transition checkpoint before as well as after DNA damage. STEM CELLS2012;30:1362–1372

Missense Mutations Located in Structural p53 DNA-Binding Motifs Are Associated With Extremely Poor Survival in Chronic Lymphocytic Leukemia

PURPOSE There is a distinct connection between TP53 defects and poor prognosis in chronic lymphocytic leukemia (CLL). It remains unclear whether patients harboring TP53 mutations represent a homogenous prognostic group. PATIENTS AND METHODS We evaluated the survival of patients with CLL and p53 defects identified at our institution by p53 yeast functional assay and complementary interphase fluorescence in situ hybridization analysis detecting del(17p) from 2003 to 2010. RESULTS A defect of the TP53 gene was identified in 100 of 550 patients. p53 mutations were strongly associated with the deletion of 17p and the unmutated IgVH locus (both P < .001). Survival assessed from the time of abnormality detection was significantly reduced in patients with both missense (P < .001) and nonmissense p53 mutations (P = .004). In addition, patients harboring missense mutation located in p53 DNA-binding motifs (DBMs), structurally well-defined parts of the DNA-binding domain, manifested a clearly shorter median survival (12 months) compared with patients having missense mutations outside DBMs (41 months; P = .002) or nonmissense alterations (36 months; P = .005). The difference in survival was similar in the analysis limited to patients harboring mutation accompanied by del(17p) and was also confirmed in a subgroup harboring TP53 defect at diagnosis. The patients with p53 DBMs mutation (at diagnosis) also manifested a short median time to first therapy (TTFT; 1 month). CONCLUSION The substantially worse survival and the short TTFT suggest a strong mutated p53 gain-of-function phenotype in patients with CLL with DBMs mutations. The impact of p53 DBMs mutations on prognosis and response to therapy should be analyzed in investigative clinical trials.

B‐cell receptor signalling and its crosstalk with other pathways in normal and malignant cells

The physiology of B cells is intimately connected with the function of their B‐cell receptor (BCR). B‐cell lymphomas frequently (dys)regulate BCR signalling and thus take advantage of this pre‐existing pathway for B‐cell proliferation and survival. This has recently been underscored by clinical trials demonstrating that small molecules (fosfamatinib, ibrutinib, idelalisib) inhibiting BCR‐associated kinases (SYK, BTK, PI3K) have an encouraging clinical effect. Here we describe the current knowledge of the specific aspects of BCR signalling in diffuse large B‐cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukaemia (CLL) and normal B cells. Multiple factors can contribute to BCR pathway (dys)regulation in these malignancies and the activation of ‘chronic’ or ‘tonic’ BCR signalling. In lymphoma B cells, the balance of initiation, amplitude and duration of BCR activation can be influenced by a specific immunoglobulin structure, the expression and mutations of adaptor molecules (like GAB1, BLNK, GRB2, CARD11), the activity of kinases (like LYN, SYK, PI3K) or phosphatases (like SHIP‐1, SHP‐1 and PTEN) and levels of microRNAs. We also discuss the crosstalk of BCR with other signalling pathways (NF‐κB, adhesion through integrins, migration and chemokine signalling) to emphasise that the ‘BCR inhibitors’ target multiple pathways interconnected with BCR, which might explain some of their clinical activity.

MicroRNA-650 expression is influenced by immunoglobulin gene rearrangement and affects the biology of chronic lymphocytic leukemia

MicroRNAs (miRNAs) play a key role in chronic lymphocytic leukemia as well as in normal B cells. Notably, miRNA gene encoding miR-650 and its homologs overlap with several variable (V) subgenes coding for lambda immunoglobulin (IgLλ). Recent studies describe the role of miR-650 in solid tumors, but its role in chronic lymphocytic leukemia (CLL) has not yet been studied. Our experiments demonstrate that miR-650 expression is regulated by coupled expression with its host gene for IgLλ. This coupling provides a unique yet unobserved mechanism for microRNA gene regulation. We determine that higher expression of miR-650 is associated with a favorable CLL prognosis and influences the proliferation capacity of B cells. We also establish that in B cells, miR-650 targets proteins important in cell proliferation and survival: cyclin dependent kinase 1 (CDK1), inhibitor of growth 4 (ING4), and early B-cell factor 3 (EBF3). This study underscores the importance of miR-650 in CLL biology and normal B-cell physiology.

MicroRNAs in chronic lymphocytic leukemia: from causality to associations and back

MicroRNAs in chronic lymphocytic leukemia: from causality to associations and back, MicroRNAs in chronic lymphocytic leukemia: from causality to associations and back