Non-Coding RNAs in Cancer Radiosensitivity:MicroRNAs and lncRNAs as Regulators of Radiation-Induced Signaling Pathways

Radiotherapy is a cancer treatment that applies high doses of ionizing radiation to induce cell death, mainly by triggering DNA double-strand breaks. The outcome of radiotherapy greatly depends on radiosensitivity of cancer cells, which is determined by multiple proteins and cellular processes. In this review, we summarize current knowledge on the role of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), in determining the response to radiation. Non-coding RNAs modulate ionizing radiation response by targeting key signaling pathways, including DNA damage repair, apoptosis, glycolysis, cell cycle arrest, and autophagy. Additionally, we indicate miRNAs and lncRNAs that upon overexpression or inhibition alter cellular radiosensitivity. Current data indicate the potential of using specific non-coding RNAs as modulators of cellular radiosensitivity to improve outcome of radiotherapy

MiR-378a-3p Is Critical for Burkitt Lymphoma Cell Growth

Simple Summary MicroRNAs (miRNAs) are small RNAs that regulate expression of specific target genes. We observed elevated levels of miR-378a-3p in Burkitt lymphoma (BL) and studied its role in the pathogenesis of BL. Inhibition of miR-378a-3p reduced growth of BL cells, confirming its significance in BL. Identification of BL specific target genes of miR-378a-3p revealed four candidates. For two of them, MNT and IRAK4, miR-378a-dependent regulation was confirmed at the protein level. Overexpression of MNT and IRAK4 in BL cell lines resulted in a similar effect as observed upon miR-378a-3p inhibition, suggesting their involvement in the growth regulatory role of miR-378a-3p. MicroRNAs (miRNAs) are small RNA molecules with important gene regulatory roles in normal and pathophysiological cellular processes. Burkitt lymphoma (BL) is an MYC-driven lymphoma of germinal center B (GC-B) cell origin. To gain further knowledge on the role of miRNAs in the pathogenesis of BL, we performed small RNA sequencing in BL cell lines and normal GC-B cells. This revealed 26 miRNAs with significantly different expression levels. For five miRNAs, the differential expression pattern was confirmed in primary BL tissues compared to GC-B cells. MiR-378a-3p was upregulated in BL, and its inhibition reduced the growth of multiple BL cell lines. RNA immunoprecipitation of Argonaute 2 followed by microarray analysis (Ago2-RIP-Chip) upon inhibition and ectopic overexpression of miR-378a-3p revealed 63 and 20 putative miR-378a-3p targets, respectively. Effective targeting by miR-378a-3p was confirmed by luciferase reporter assays for MAX Network Transcriptional Repressor (MNT), Forkhead Box P1 (FOXP1), Interleukin 1 Receptor Associated Kinase 4 (IRAK4), and lncRNA Just Proximal To XIST (JPX), and by Western blot for IRAK4 and MNT. Overexpression of IRAK4 and MNT phenocopied the effect of miR-378a-3p inhibition. In summary, we identified miR-378a-3p as a miRNA with an oncogenic role in BL and identified IRAK4 and MNT as miR-378a-3p target genes that are involved in its growth regulatory role

Inhibition of the miR-155 target NIAM phenocopies the growth promoting effect of miR-155 in B-cell lymphoma

Several studies have indicated an important role for miR-155 in the pathogenesis of B-cell lymphoma. Highly elevated levels of miR-155 were indeed observed in most B-cell lymphomas with the exception of Burkitt lymphoma (BL). However, the molecular mechanisms that underlie the oncogenic role of miR-155 in B-cell lymphoma are not well understood. To identify the miR-155 targets relevant for B-cell lymphoma, we performed RNA immunoprecipitation of Argonaute 2 in Hodgkin lymphoma (HL) cells upon miR-155 inhibition and in BL cells upon ectopic expression of miR-155. We identified 54 miR-155-specific target genes in BL cells and confirmed miR-155 targeting of DET1, NIAM, TRIM32, HOMEZ, PSIP1 and JARID2. Five of these targets are also regulated by endogenous miR-155 in HL cells. Both overexpression of miR-155 and inhibition of expression of the novel miR-155 target gene NIAM increased proliferation of BL cells. In primary B-cell lymphoma NIAM-positive cases have significant lower levels of miR-155 as compared to NIAM-negative cases, suggesting that NIAM is also regulated by miR-155 in primary B-cell lymphoma. Thus, our data indicate an oncogenic role for miR-155 in B-cell lymphoma which involves targeting the tumor suppressor NIAM

Argonaute 2 immunoprecipitation revealed large tumor suppressor kinase 1 as a novel proapoptotic target of miR-21 in T cells

MicroRNA (miR)-21 is an important suppressor of T-cell apoptosis that is also overexpressed in many types of cancers. The exact mechanisms underlying the antiapoptotic effects of miR-21 are not well understood. In this study, we used the Jurkat T-cell line as a model to identify apoptosis-associated miR-21 target genes. We showed that expression of miR-21 rapidly increases upon alpha CD3/alpha CD28 activation of Jurkat cells. Inhibition of miR-21 reduced cell growth which could be explained by an increase in apoptosis. MicroRNA target gene identification by AGO2 RNA-immunoprecipitation followed by gene expression microarray (RIP-Chip) resulted in the identification of 72 predicted miR-21 target genes that were at least twofold enriched in the AGO2-IP fraction of miR-21 overexpressing cells. Of these, 71 were at least twofold more enriched in the AGO2-IP fraction of miR-21 overexpressing cells as compared to AGO2-IP fraction of control cells. The target gene for which the AGO2-IP enrichment was most prominently increased upon miR-21 overexpression was the proapoptotic protein LATS1. Luciferase reporter assays and western blot analysis confirmed targeting of LATS1 by miR-21. qRT-PCR analysis in primary T cells showed an inverse expression pattern between LATS1 transcript levels and miR-21 upon T-cell stimulation. Finally, LATS1 knockdown partially rescued the miR-21 inhibition-induced impaired cell growth. Collectively, these data identify LATS1 as a miR-21 target important for the antiapoptotic function of miR-21 in T cells and likely also in many types of cancer

Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies

Causes and consequences of altered microRNA levels : regulation of microRNA biogenesis and identification of microRNA-155 target genes

Micro(mi)RNAs reguleren de expressie van een groot aantal genen en zijn daardoor betrokken bij de regulatie van bijna alle cellulaire processen. Veranderdingen in het miRNA expressie patroon is kenmerkend voor kankercellen in het algemeen en wordt ook gevonden bij B cel lymfomen. Het doel van deze studie is om te onderzoeken welke mechanismen bijdragen aan het veranderde miRNA expressie profiel en meer specifiek wat de gevolgen zijn van een veranderd miR-155 expressie nivo. We hebben de biogenese onderzocht van drie miRNAs die liggen in exonen van niet voor eiwit coderende genen. De focus was op de subcellulaire locatie van de primaire transcripten voor en na het verwijderen van de intronen. Daarnaast hebben we aangetoond dat het expressie patroon van zes miRNAs, afkomstig van het bekende oncogene miR-17~92 cluster, onderling sterk verschilden in normale B cel subsets en ook in vier veel voorkomende B cel lymfoom subtypen. In vergelijking met de normale B cel voorloper cellen bleek dat in alle vier lymfoom subtypen miR-19b het sterkste verhoogd was. Tot slot hebben we de functie van miR-155 onderzocht door de expressie van dit miRNA experimenteel te moduleren. Over expressie van miR-155 in een B cel lymfoom subtype met een lage endogene miR-155 expressie gaf een groei voordeel aan de tumor cellen, en dit effect kon worden verklaard door effectieve binding van miR-155 aan het TBRG1 transcript resulterend in een verlaagde TBRG1 expressie. MiRNAs are effective gene expression regulators that play crucial roles in most cellular processes. Altered miRNA levels are observed in almost all types of B-cell lymphoma. The aim of this thesis is to investigate possible causes of altered miRNA levels and the consequences of altered miRNA-155 levels in B-cell lymphoma. We present an overview of established mechanisms to regulate miRNA processing (chapter 2). We next studied processing of a relatively small group of miRNAs that are located in exons of noncoding RNAs (chapter 3). Based on subcellular location and overexpression experiments we concluded that miRNAs are processed predominantly from the unspliced transcripts that almost completely reside in the nucleus. In chapter 4, we studied processing of the oncogenic miR-17~92 cluster in normal B cell subsets and in four B-cell non-Hodgkin lymphoma (NHL) subtypes. We observed marked differences between the levels of the six miRNAs in the B cell subsets and the NHL subtype. In comparison to their normal counterparts all lymphoma subtypes showed a marked induction of miR-19b. In chapter 5, we describe a straight-forward method to generate viral miRNA sponge constructs that can be used to inhibit miRNA functioning. In chapter 6, we studied the consequence of altered miR-155 expression levels in B-cell lymphoma. Induction of miR-155 revealed a significant growth advantage of the tumor cells. Using Ago2-RIP-Chip and phenotype copy experiments we showed that TBRG1 is one of the miR-155 targets. Inhibition of TBRG1 also induced a growth advantage similar to the miR-155 effect.

Conditions of hydrodynamic instability appearance in fluid thin layers with changes in time thickness and density gradient

The transport of NaCl and ethanol through the microbial cellulose membrane was used as a generator of layers with density gradients, parallel to gravity vector. Changes in NaCl concentrations connected with density gradients in these layers were monitored by means of Ag vertical bar AgCl electrodes dipped directly into aqueous NaCl solutions with or without ethanol. The parameters used in this article and characterizing diffusive or diffusive convective processes in layers are the ratio of NaCl concentrations at electrode surfaces (C-i*/C-e*) calculated for voltage which was measured 6h after rebuilding of layers with density gradients, and time of appearance of hydrodynamic instabilities in the membrane system. The C-i*/C-e* is the nonlinear function of initial ratio of NaCl on the membrane (C-h/C-l), with maximum concentration at initial moment dependent on ethanol and configuration of the membrane system. The time of appearance of hydrodynamic instabilities in layers with density gradients parallel and directed opposite to the gravity vector depends on C-h/C-l, ethanol concentration, and configuration of the membrane system. Besides, for aqueous NaCl solutions, critical values of density gradients and thicknesses of layers needed for the appearance of hydrodynamic instabilities were calculated and presented as functions of C-h/C-l

Micro RNAs in Regulation of Cellular Redox Homeostasis

In living cells Reactive Oxygen Species (ROS) participate in intra- and inter-cellular signaling and all cells contain specific systems that guard redox homeostasis. These systems contain both enzymes which may produce ROS such as NADPH-dependent and other oxidases or nitric oxide synthases, and ROS-neutralizing enzymes such as catalase, peroxiredoxins, thioredoxins, thioredoxin reductases, glutathione reductases, and many others. Most of the genes coding for these enzymes contain sequences targeted by micro RNAs (miRNAs), which are components of RNA-induced silencing complexes and play important roles in inhibiting translation of their targeted messenger RNAs (mRNAs). In this review we describe miRNAs that directly target and can influence enzymes responsible for scavenging of ROS and their possible role in cellular redox homeostasis. Regulation of antioxidant enzymes aims to adjust cells to survive in unstable oxidative environments; however, sometimes seemingly paradoxical phenomena appear where oxidative stress induces an increase in the levels of miRNAs which target genes which are supposed to neutralize ROS and therefore would be expected to decrease antioxidant levels. Here we show examples of such cellular behaviors and discuss the possible roles of miRNAs in redox regulatory circuits and further cell responses to stress